Use of truncated cysteine il28 and il29 mutants to treat cancers and autoimmune disorders

ABSTRACT

Methods for treating patients with cancer and autoimmune disorders using IL-28 and IL-29 molecules. The IL-28 and IL-29 molecules include polypeptides that have homology to the human IL-28 or IL-29 polypeptide sequence and proteins fused to a polypeptide with IL-28 and IL-29 functional activity. The molecules can be used as a monotherapy or in combination with other known cancer and/or autoimmune therapeutics.

CROSS-REFERENCE TO RELATED APPLICATIONS

This present application is a continuation of U.S. patent applicationSer. No. 11/489,894, filed Jul. 20, 2006, which claims the benefit ofU.S. Patent Application Ser. Nos. 60/771,260, filed Feb. 8, 2006, and60/700,951, filed Jul. 20, 2005, all of which are herein incorporated byreference.

BACKGROUND OF THE INVENTION

Cytokines generally stimulate proliferation or differentiation of cellsof the hematopoietic lineage or participate in the immune andinflammatory response mechanisms of the body. Examples of cytokineswhich affect hematopoiesis are erythropoietin (EPO), which stimulatesthe development of red blood cells; thrombopoietin (TPO), whichstimulates development of cells of the megakaryocyte lineage; andgranulocyte-colony stimulating factor (G-CSF), which stimulatesdevelopment of neutrophils. These cytokines are useful in restoringnormal blood cell levels in patients suffering from anemia,thrombocytopenia, and neutropenia or receiving chemotherapy for cancer.

The interleukins are a family of cytokines that mediate immunologicalresponses. Central to an immune response is the T cell, which producemany cytokines and adaptive immunity to antigens. Cytokines produced bythe T cell have been classified as type 1 and type 2 (Kelso, A. Immun.Cell Biol. 76:300-317, 1998). Type 1 cytokines include IL-2, IFN-γ,LT-α, and are involved in inflammatory responses, viral immunity,intracellular parasite immunity and allograft rejection. Type 2cytokines include IL-4, IL-5, IL-6, IL-10 and IL-13, and are involved inhumoral responses, helminth immunity and allergic response. Sharedcytokines between Type 1 and 2 include IL-3, GM-CSF and TNF-α. There issome evidence to suggest that Type 1 and Type 2 producing T cellpopulations preferentially migrate into different types of inflamedtissue.

The immune system is the body's primary defense against diseases causedby pathogens, namely bacteria, viruses, fungi etc, as well as againstdiseases caused by abnormal growth of the body's own cells and tissues(i.e. cancerous tumors). Normally, the immune system is able todistinguish between the body's normal cells or “self” and foreignpathogens or abnormal cells or “non-self”. The processes by which theimmune system refrains from reacting to one's own body is calledtolerance. Sometimes, the immune system loses the ability to recognize“self” as normal and the subsequent response directed against the tissueor cells, results in loss of tolerance, a state of autoimmunity. Thepathologies resulting from autoimmunity often have serious clinicalconsequences and are one of the major health problems in the world,especially in developed nations.

One example of such an autoimmune disorder is multiple sclerosis (MS), aprogressive disease of the central nervous system (CNS). In MS patients,the patient's own immune system destroys myelin, the protective layerthat surrounds and insulates the nerve fibers in the brain and spinalcord. The destruction of the myelin sheath leads to disruption ofneurotransmission and scarring damage to the nerve fibers. The endresult is the manifestation of numerous symptoms in the affected patientincluding tingling or numbness, slurred speech, impaired vision, vertigoetc. Over the course of the disease, there is loss of strength in theextremities, leading to problems with movement and in the most severecases, leading to paralysis of the limbs. Based on clinical diagnosis,there are currently four types of MS classifications, based on whichpart of the brain or spinal cord are affected, severity, frequency ofattacks etc.

Current therapies for MS include corticosteroid drugs (to alleviatesymptoms of acute episodes), as well as other drugs like IFN-β andNovantrone®. Novantrone® has been approved for late stage MS patients,specifically for whom other therapies have not worked. Novantrone® iscytotoxic to most cells and therefore as one would expect, has an arrayof side effects and is toxic at doses required for the maximaltherapeutic effects. IFN-β is also toxic, limiting dosage of the drug inMS patients. Furthermore, continuous use of these drugs has been shownto desensitize patients to further use of the same drug, therebylimiting the ability to use these drugs as long term therapeutics.

Of particular interest, from a therapeutic standpoint, are theinterferons (reviews on interferons are provided by De Maeyer and DeMaeyer-Guignard, “Interferons,” in The Cytokine Handbook, 3^(rd)Edition, Thompson (ed.), pages 491-516 (Academic Press Ltd. 1998), andby Walsh, Biopharmaceuticals: Biochemistry and Biotechnology, pages158-188 (John Wiley & Sons 1998)). Interferons exhibit a variety ofbiological activities, and are useful for the treatment of certainautoimmune diseases, particular cancers, and the enhancement of theimmune response against infectious agents, including viruses, bacteria,fungi, and protozoa. To date, six forms of interferon have beenidentified, which have been classified into two major groups. Theso-called “type I” IFNs include IFN-α, IFN-β, IFN-ω, IFN-δ, andinterferon-τ. Currently, IFN-γ and one subclass of IFN-α are the onlytype II IFNs.

Type I IFNs, which are thought to be derived from the same ancestralgene, have retained sufficient similar structure to act by the same cellsurface receptor. The α-chain of the human IFN-α/β receptor comprises anextracellular N-terminal domain, which has the characteristics of aclass II cytokine receptor. IFN-γ does not share significant homologywith the type I IFN or with the type II IFN-α subtype, but shares anumber of biological activities with the type I IFN.

Clinicians are taking advantage of the multiple activities ofinterferons by using the proteins to treat a wide range of conditions.For example, one form of IFN-α has been approved for use in more than 50countries for the treatment of medical conditions such as hairy cellleukemia, renal cell carcinoma, basal cell carcinoma, malignantmelanoma, AIDS-related Kaposi's sarcoma, multiple myeloma, chronicmyelogenous leukemia, non-Hodgkin's lymphoma, laryngeal papillomatosis,mycosis fungoides, condyloma acuminata, chronic hepatitis B, hepatitisC, chronic hepatitis D, and chronic non-A, non-B/C hepatitis. The U.S.Food and Drug Administration has approved the use of IFN-β to treatmultiple sclerosis, a chronic disease of the nervous system. IFN-γ isused to treat chronic granulomatous diseases, in which the interferonenhances the patient's immune response to destroy infectious bacterial,fungal, and protozoal pathogens. Clinical studies also indicate thatIFN-γ may be useful in the treatment of AIDS, leishmaniasis, andlepromatous leprosy.

IL-28A, IL-28B, and IL-29 comprise a recently discovered new family ofproteins that have sequence homology to type I interferons and genomichomology to IL-10. This new family is fully described in co-owned PCTapplication WO 02/086087 and Sheppard et al., Nature Immunol. 4:63-68,2003; both incorporated by reference herein. Functionally, IL-28 andIL-29 resemble type I INFs in their ability to induce an antiviral statein cells but, unlike type I IFNs, they do not display antiproliferativeactivity against certain B cell lines.

Mature T cells can be activated, i.e., by an antigen or other stimulus,to produce, for example, cytokines, biochemical signaling molecules, orreceptors that further influence the fate of the T cell population.

B cells can be activated via receptors on their cell surface including Bcell receptor and other accessory molecules to perform accessory cellfunctions, such as production of cytokines. B cell activation results inthe production of antibodies that can bind to immunogenic cell-surfaceproteins on tumor cells and initiate complement-mediated cell lysis,bridge NK cells or macrophages to the tumor for antibody-dependentcell-mediated cytotoxicity (ADCC), interfere with tumor cell growth byblocking survival or inducing apoptotic signals, or increaseimmunogenicity by facilitating the uptake and presentation of tumorantigens by APCs. Thus, enhancing B cell responses in vivo has thepotential to promote antitumor activity (Blattman et al., Science,305:200-205 (Jul. 9, 2004)).

Therefore, agents which can augment natural host defenses against tumorinduction or progression may increase remission rates and enhancesurvival of patients, without the cytotoxic side effects of priormethods.

The present invention provides such methods for treating solid tumors,lymphomas, and autoimmune disorders by administrating IL-28A, IL-28B, orIL-29 compositions that may be used as a monotherapy or in combinationwith chemotherapy, radiation therapy, small molecules or otherbiologics. These and other uses should be apparent to those skilled inthe art from the teachings herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows mice injected with mouse IL-28 plasmid on Days 5 and 12inhibit RENCA tumor growth in vivo.

FIG. 2 shows mice injected with mouse IL-28 plasmid, mouse IFN-αplasmid, and human IL-29 C172S polypeptide N-terminally conjugated to a20 kD methoxy-polyethylene glycol propionaldehyde inhibit RENCA tumorgrowth in vivo. Plasmid injections are on Days 5 and 12. Protein wasgiven every other day from day 5-21.

FIG. 3 shows mice injected with 1 μg, 5 μg and 25 μg of human IL-29C172S polypeptide N-terminally conjugated to a 20 kDmethoxy-polyethylene glycol propionaldehyde and human IL-29 C172S d2-7polypeptide N-terminally conjugated to a 20 kD methoxy-polyethyleneglycol propionaldehyde inhibit RENCA tumor growth in vivo. All proteingiven every other day from days 5-23.

FIG. 4 shows mice injected with vehicle (▪), 5 μg human IL-29 C172S d2-7polypeptide N-terminally conjugated to a 20 kD methoxy-polyethyleneglycol propionaldehyde (▾), and 25 μg human IL-29 C172S d2-7 polypeptideN-terminally conjugated to a 20 kD methoxy-polyethylene glycolpropionaldehyde (♦) every-other-day for 20 days once tumor volumereached 100 mm³, 5 μg human IL-29 C172S d2-7 polypeptide N-terminallyconjugated to a 20 kD methoxy-polyethylene glycol propionaldehydeeveryday for 20 days once tumor volume reached 100 mm³ (), and 5 μghuman IL-29 C172S d2-7 polypeptide N-terminally conjugated to a 20 kDmethoxy-polyethylene glycol propionaldehyde administeredprophylactically every other day for 20 days starting on day 5 of tumorinjection (▴).

FIG. 5A shows mice injected with 25 μg human IL-29 C172S d2-7polypeptide N-terminally conjugated to a 20 kD methoxy-polyethyleneglycol propionaldehyde or vehicle beginning on Day 0 and ten subsequenti.p. injections every-other-day prolongs survival of the mice in theE.G7 thymoma model.

FIG. 5B shows mice injected with 25 μg human IL-29 C172S d2-7polypeptide N-terminally conjugated to a 20 kD methoxy-polyethyleneglycol propionaldehyde or vehicle beginning on Day 0 and ten subsequenti.p. injections every-other-day inhibits tumor growth in the E.G7thymoma model.

FIG. 6 shows the prophylactic administration of PEG-mIL-28 inhibitsdisease severity and incidence in RR-EAE model.

FIG. 7 shows the prophylactic administration of PEG-mIL-28 significantlyreduces incidence of clinical disease in mice with RR-EAE.

FIG. 8 shows the prophylactic administration of PEG-rIL-29 inhibitsdisease severity and incidence in the RR-EAE mouse model.

FIG. 9 shows the prophylactic administration of PEG-rIL-29 significantlyreduces incidence of clinical disease in mice with RR-EAE.

FIG. 10 shows the therapeutic administration with PEG-mIL-28significantly inhibits disease severity in RR-EAE model.

FIG. 11 shows the therapeutic administration of PEG-mIL-28 reducesclinical severity in the RR-EAE model. Average total clinical score forthe study period are shown.

FIG. 12 shows the therapeutic administration of PEG-mIL-28 increasepercent of mice with complete remission in the RR-EAE model.

DESCRIPTION OF THE INVENTION

In the description that follows, a number of terms are used extensively.The following definitions are provided to facilitate understanding ofthe invention.

Unless otherwise specified, “a,” “an,” “the,” and “at least one” areused interchangeably and mean one or more than one.

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase(Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985),substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-10, 1988),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2: 95-107, 1991. DNAs encoding affinity tags are availablefrom commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

The term “allelic variant” is used herein to denote any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inphenotypic polymorphism within populations. Gene mutations can be silent(no change in the encoded polypeptide) or may encode polypeptides havingaltered amino acid sequence. The term allelic variant is also usedherein to denote a protein encoded by an allelic variant of a gene.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “cancer” or “cancer cell” is used herein to denote a tissue orcell found in a neoplasm which possesses characteristics whichdifferentiate it from normal tissue or tissue cells. Among suchcharacteristics include but are not limited to: degree of anaplasia,irregularity in shape, indistinctness of cell outline, nuclear size,changes in structure of nucleus or cytoplasm, other phenotypic changes,presence of cellular proteins indicative of a cancerous or pre-cancerousstate, increased number of mitoses, and ability to metastasize. Wordspertaining to “cancer” include carcinoma, sarcoma, tumor, epithelioma,leukemia, lymphoma, polyp, and scirrus, transformation, neoplasm, andthe like.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity of<10⁹ M⁻¹.

The term “complements of a polynucleotide molecule” denotes apolynucleotide molecule having a complementary base sequence and reverseorientation as compared to a reference sequence.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons (as compared toa reference polynucleotide molecule that encodes a polypeptide).Degenerate codons contain different triplets of nucleotides, but encodethe same amino acid residue (i.e., GAU and GAC triplets each encodeAsp).

The term “expression vector” is used to denote a DNA molecule, linear orcircular, that comprises a segment encoding a polypeptide of interestoperably linked to additional segments that provide for itstranscription. Such additional segments include promoter and terminatorsequences, and may also include one or more origins of replication, oneor more selectable markers, an enhancer, a polyadenylation signal, etc.Expression vectors are generally derived from plasmid or viral DNA, ormay contain elements of both.

The term “isolated”, when applied to a polynucleotide, denotes that thepolynucleotide has been removed from its natural genetic milieu and isthus free of other extraneous or unwanted coding sequences, and is in aform suitable for use within genetically engineered protein productionsystems. Such isolated molecules are those that are separated from theirnatural environment and include cDNA and genomic clones. Isolated DNAmolecules of the present invention are free of other genes with whichthey are ordinarily associated, but may include naturally occurring 5′and 3′ untranslated regions such as promoters and terminators. Theidentification of associated regions will be evident to one of ordinaryskill in the art (see for example, Dynan and Tijan, Nature 316:774-78,1985).

An “isolated” polypeptide or protein is a polypeptide or protein that isfound in a condition other than its native environment, such as apartfrom blood and animal tissue. In a preferred form, the isolatedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin. It is preferred to provide thepolypeptides in a highly purified form, i.e. greater than 95% pure, morepreferably greater than 99% pure. When used in this context, the term“isolated” does not exclude the presence of the same polypeptide inalternative physical forms, such as dimers or alternatively glycosylatedor derivatized forms.

The term “level” when referring to immune cells, such as NK cells, Tcells, in particular cytotoxic T cells, B cells and the like, anincreased level is either increased number of cells or enhanced activityof cell function.

The term “neoplastic”, when referring to cells, indicates cellsundergoing new and abnormal proliferation, particularly in a tissuewhere in the proliferation is uncontrolled and progressive, resulting ina neoplasm. The neoplastic cells can be either malignant, i.e. invasiveand metastatic, or benign.

The term “operably linked”, when referring to DNA segments, indicatesthat the segments are arranged so that they function in concert fortheir intended purposes, e.g., transcription initiates in the promoterand proceeds through the coding segment to the terminator.

A “polynucleotide” is a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′end. Polynucleotides include RNA and DNA, and may be isolated fromnatural sources, synthesized in vitro, or prepared from a combination ofnatural and synthetic molecules. Sizes of polynucleotides are expressedas base pairs (abbreviated “bp”), nucleotides (“nt”), or kilobases(“kb”). Where the context allows, the latter two terms may describepolynucleotides that are single-stranded or double-stranded. When theterm is applied to double-stranded molecules it is used to denoteoverall length and will be understood to be equivalent to the term “basepairs”. It will be recognized by those skilled in the art that the twostrands of a double-stranded polynucleotide may differ slightly inlength and that the ends thereof may be staggered as a result ofenzymatic cleavage; thus all nucleotides within a double-strandedpolynucleotide molecule may not be paired.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides”.

The term “promoter” is used herein for its art-recognized meaning todenote a portion of a gene containing DNA sequences that provide for thebinding of RNA polymerase and initiation of transcription. Promotersequences are commonly, but not always, found in the 5′ non-codingregions of genes.

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule (i.e., a ligand) and mediates the effect of theligand on the cell. Membrane-bound receptors are characterized by amulti-peptide structure comprising an extracellular ligand-bindingdomain and an intracellular effector domain that is typically involvedin signal transduction. Binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell. This interactionin turn leads to an alteration in the metabolism of the cell. Metabolicevents that are linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids. In general, receptors can be membranebound, cytosolic or nuclear; monomeric (e.g., thyroid stimulatinghormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGFreceptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSFreceptor, erythropoietin receptor and IL-6 receptor).

The term “secretory signal sequence” denotes a DNA sequence that encodesa polypeptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

Molecular weights and lengths of polymers determined by impreciseanalytical methods (e.g., gel electrophoresis) will be understood to beapproximate values. When such a value is expressed as “about” X or“approximately” X, the stated value of X will be understood to beaccurate to ±10%.

“zcyto20”, “zcyto21”, “zcyto22” are the previous designations for humanIL-28A, human IL-29, and human IL-28B, respectively, and are usedinterchangeably herein. The nucleotide and amino acid sequence forIL-28A are shown in SEQ ID NO:1 and SEQ ID NO:2, respectively. Thenucleotide and amino acid sequences for IL-29 are shown in SEQ ID NO:3and SEQ ID NO:4, respectively. The nucleotide and amino acid sequencefor IL-28B are shown in SEQ ID NO:5 and SEQ ID NO:6, respectively. Thesesequences are fully described in PCT application WO 02/086087, U.S. Pat.No. 6,927,040, PCT Application WO 04/037995, PCT Application WO05/023862, PCT Application WO 05/097165, and PCT Application 06/012644commonly assigned to ZymoGenetics, Inc., all of which are hereinincorporated by reference.

“zcyto24” and “zcyto25” are the previous designations for mouse IL-28,and are shown in SEQ ID NOs: 7, 8, 9, 10, respectively. Thepolynucleotide and polypeptides are fully described in PCT applicationWO 02/086087 commonly assigned to ZymoGenetics, Inc., incorporatedherein by reference.

“zcytor19” is the previous designation for IL-28 receptor α-subunit, andis shown in SEQ ID NO: 11. The polynucleotides and polypeptides aredescribed in PCT application WO 02/20569 on behalf of Schering, Inc.,and WO 02/44209 assigned to ZymoGenetics, Inc and incorporated herein byreference. “IL-28 receptor” denotes the IL-28 α-subunit and CRF2-4subunit forming a heterodimeric receptor.

All references cited herein are incorporated by reference in theirentirety.

A. IL-28, IL-29 and its Receptor

When referring to IL-28, the term shall mean both IL-28A and IL-28B.Previously IL-28A was designated zcyto20 (SEQ ID NOs: 1 and 2), IL-29was designated zcyto21 (SEQ ID NOs: 3 and 4), and IL-28B was designatedzcyto22 (SEQ ID NOs:5 and 6). (See, PCT application WO 02/086087 andSheppard et al., supra.) The mouse orthologs for IL-28 were previouslydesignated as zcyto24 (SEQ ID NOs:7 and 8), zcyto25 (SEQ ID NOs: 9 and10).

Wildtype IL-28A gene encodes a polypeptide of 200 amino acids, as shownin SEQ ID NO:2. The signal sequence for IL-28A can be predicted ascomprising amino acid residue-25 (Met) through amino acid residue-1(Ala) of SEQ ID NO:2. The mature peptide for IL-28A begins at amino acidresidue 1 (Val). IL-28A helices are predicted as follow: helix A isdefined by amino acid residues 24 (Leu) to 40 (Glu); helix B by aminoacid residues 58 (Thr) to 65 (Gln); helix C by amino acid residues 69(Arg) to 85 (Ala); helix D by amino acid residues 95 (Val) to 114 (Ala);helix E by amino acid residues 126 (Thr) to 142 (Lys); and helix F byamino acid residues 148 (Cys) to 169 (Ala); as shown in SEQ ID NO: 2.

Wildtype IL-29 gene encodes a polypeptide of 200 amino acids, as shownin SEQ ID NO:4. The signal sequence for IL-29 can be predicted ascomprising amino acid residue-19 (Met) through amino acid residue-1(Ala) of SEQ ID NO:4, SEQ ID NO:119, or SEQ ID NO:121. The maturepeptide for IL-29 begins at amino acid residue 1 (Gly). IL-29 has beendescribed in PCT application WO 02/02627. IL-29 helices are predicted asfollows: helix A is defined by amino acid residues 30 (Ser) to 44 (Leu);helix B by amino acid residues 57 (Asn) to 65 (Val); helix C by aminoacid residues 70(Val) to 85 (Ala); helix D by amino acid residues 92(Glu) to 114 (Gln); helix E by amino acid residues 118 (Thr) to 139(Lys); and helix F by amino acid residues 144 (Gly) to 170 (Leu); asshown in SEQ ID NO: 4.

Wildtype IL-28B gene encodes a polypeptide of 200 amino acids, as shownin SEQ ID NO:6. The signal sequence for IL-28B can be predicted ascomprising amino acid residue-21 (Met) through amino acid residue-1(Ala) of SEQ ID NO:6. The mature peptide for IL-28B begins at amino acidresidue 1 (Val). IL-28B helices are predicted as follow: helix A isdefined by amino acid residues 8 (Leu) to 41 (Glu); helix B by aminoacid residues 58 (Trp) to 65 (Gln); helix C by amino acid residues 69(Arg) to 86 (Ala); helix D by amino acid residues 95 (Gly) to 114 (Ala);helix E by amino acid residues 126 (Thr) to 142 (Lys); and helix F byamino acid residues 148 (Cys) to 169 (Ala); as shown in SEQ ID NO: 6.

The present invention provides mutations in the IL-28 and IL-29 wildtypesequences as shown in, for example, SEQ ID NOs: 1, 2, 3, 4, 5, and 6,that result in expression of single forms of the IL-28 or IL-29molecule. Because the heterogeneity of forms is believed to be a resultof multiple intramolecular disulfide bonding patterns, specificembodiments of the present invention includes mutations to the cysteineresidues within the wildtype IL-28 and IL-29 sequences. When IL-28 andIL-29 are expressed in E. coli, an N-terminal Methionine is present. SEQID NOs:12-17, for example, show the nucleotide and amino acid residuenumbering for IL-28A, IL-29 and IL-28B when the N-terminal Met ispresent. Table 1 shows the possible combinations of intramoleculardisulfide bonded cysteine pairs for wildtype IL-28A, IL-28B, and IL-29.

TABLE 1 IL-28A C₁₆-C₁₁₅ C₄₈-C₁₄₈ C₅₀-C₁₄₈ C₁₆₇-C₁₇₄ C₁₆-C₄₈ C₁₆-C₅₀C₄₈-C₁₁₅ C₅₀-C₁₁₅ C₁₁₅-C₁₄₈ SEQ ID NO: 2 Met IL- C₁₇-C₁₁₆ C₄₉-C₁₄₉C₅₁-C₁₄₉₈ C₁₆₈-C₁₇₅ C₁₇-C₄₉ C₁₇-C₅₁ C₄₉-C₁₁₆ C₅₁-C₁₁₆ C₁₁₆-C₁₄₉ 28A SEQID NO: 13 IL-29 C₁₅-C₁₁₂ C₄₉-C₁₄₅ C₁₁₂-C₁₇₁ SEQ ID NO: 4 Met IL-29C₁₆-C₁₁₃ C₅₀-C₁₄₆ C₁₁₃-C₁₇₂ SEQ ID NO: 15 IL-28B C₁₆-C₁₁₅ C₄₈-C₁₄₈C₅₀-C₁₄₈ C₁₆₇-C₁₇₄ C₁₆-C₄₈ C₁₆-C₅₀ C₄₈-C₁₁₅ C₅₀-C₁₁₅ C₁₁₅-C₁₄₈ SEQ IDNO: 6 Met IL- C₁₇-C₁₁₆ C₄₉-C₁₄₉ C₅₁-C₁₄₉₈ C₁₆₈-C₁₇₅ C₁₇-C₄₉ C₁₇-C₅₁C₄₉-C₁₁₆ C₅₁-C₁₁₆ C₁₁₆-C₁₄₉ 28B SEQ ID NO: 17

The polynucleotide and polypeptide molecules of the present inventionmay have a mutation at one or more of the Cysteines present in thewildtype IL-28A, IL-29 or IL-28B molecules, yet retain some biologicalactivity as described herein. Table 2 illustrates exemplary Cysteinemutants, in particular point mutations of cysteine (C) to serine (S).

TABLE 2 IL-28A C48S SEQ ID NO: 19 Met IL-28A C49S SEQ ID NO: 21 IL-28AC50S SEQ ID NO: 23 Met IL-28A C51S SEQ ID NO: 25 IL-29 C171S SEQ ID NO:27 Met IL-29 C172S SEQ ID NO: 29

All the members of the family have been shown to bind to the same classII cytokine receptor, IL-28R. IL-28 α-subunit was previously designatedzcytor19 receptor. While not wanting to be bound by theory, thesemolecules appear to all signal through IL-28R receptor via the samepathway. IL-28 receptor is described in a commonly assigned PCT patentapplication WO 02/44209, incorporated by reference herein; Sheppard etal., supra; Kotenko et al., Nature Immunol 4:69-77, 2003; and PCTWO/03/040345. IL-28R is a member of the Class II cytokine receptorswhich is characterized by the presence of one or more cytokine receptormodules (CRM) in their extracellular domains. Other class II cytokinereceptors include zcytor11 (commonly owned U.S. Pat. No. 5,965,704),CRF2-4 (Genbank Accession No. Z17227), IL-10R (Genbank Accession No.sU00672 and NM_(—)001558), DIRS1, zcytor7 (commonly owned U.S. Pat. No.5,945,511), and tissue factor. IL-28 receptor, like all known class IIreceptors except interferon-alpha/beta receptor alpha chain, has only asingle class II CRM in its extracellular domain.

Four-helical bundle cytokines are also grouped by the length of theircomponent helices. “Long-helix” form cytokines generally consist ofbetween 24-30 residue helices, and include IL-6, ciliary neutrotrophicfactor (CNTF), leukemia inhibitory factor (LIF) and human growth hormone(hGH). “Short-helix” form cytokines generally consist of between 18-21residue helices and include IL-2, IL-4 and GM-CSF. Studies using CNTFand IL-6 demonstrated that a CNTF helix can be exchanged for theequivalent helix in IL-6, conferring CTNF-binding properties to thechimera. Thus, it appears that functional domains of four-helicalcytokines are determined on the basis of structural homology,irrespective of sequence identity, and can maintain functional integrityin a chimera (Kallen et al., J. Biol. Chem. 274:11859-11867, 1999).Therefore, IL-28 and IL-29 polypeptides will be useful for preparingchimeric fusion molecules, particularly with other interferons todetermine and modulate receptor binding specificity. Of particularinterest are fusion proteins that combine helical and loop domains frominterferons and cytokines such as INF-α, IL-10, human growth hormone.

The present invention provides polynucleotide molecules, including DNAand RNA molecules, which encode IL-28 or IL-29 polypeptides. Forexample, the present invention provides degenerate nucleotide sequencesencoding IL-28A C48S, Met IL-28A C49S, IL-28A C50S, Met IL-28A C51S,IL-29 C171S and Met IL-29 C172S polypeptides disclosed herein. Thoseskilled in the art will readily recognize that, in view of thedegeneracy of the genetic code, considerable sequence variation ispossible among these polynucleotide molecules. SEQ ID NOs:30, 31, 32,33, 34, and 35 are a degenerate DNA sequences that encompasses all DNAsthat encode IL-28A C48S, Met IL-28A C49S, IL-28A C50S, Met IL-28A C51S,IL-29 C171S and Met IL-29 C172S, respectively. Those skilled in the artwill recognize that the degenerate sequence of SEQ ID NOs: 30, 31, 32,33, 34, and 35 also provides all RNA sequences encoding SEQ ID NOs: 30,31, 32, 33, 34, and 35 by substituting U for T and are thus contemplatedby the present invention.

A zcyto20 or IL-28A gene encodes a polypeptide of 205 amino acids, asshown in SEQ ID NO:2. The signal sequence for IL-28A comprises aminoacid residue-25 (Met) through amino acid residue-1 (Ala) of SEQ ID NO:2,or alternatively amino acid residues-21 (Met) through amino acidresidue-1 (Ala) of SEQ ID NO:2. The mature peptide for IL-28A begins atamino acid residue 1 (Val) of SEQ ID NO:2. Zcyto20 helices are predictedas follow: helix A is defined by amino acid residues 52 (Ala) to 66(Leu); helix B by amino acid residues 78 (Arg) to 87 (Val); helix C byamino acid residues 91 (Pro) to 108 (Thr); helix D by amino acidresidues 116 (Val) to 138 (Ser); helix E by amino acid residues 151(Thr) to 172 (Lys); and helix F by amino acid residues 177 (Gly) to 197(Cys); as shown in SEQ ID NO:2. Further analysis of Zcyto20 based onmultiple alignments predicts that cysteines at amino acid residues 37and 136; 69 and 197; and 71 and 178 (as shown in SEQ ID NO:2) will formintramolecular disulfide bonds. The corresponding polynucleotidesencoding the Zcyto20 polypeptide regions, domains, motifs, residues andsequences described herein are as shown in SEQ ID NO:1. When apolynucleotide sequence encoding the mature polypeptide is expressed ina prokaryotic system, such as E. coli, the a secretory signal sequencemay not be required and the an N-terminal Met will be present, resultingin expression of a polypeptide such as is shown in SEQ ID NO:13.

IL-28A polypeptides of the present invention also include a mutation atthe second cysteine, C2, of the mature polypeptide. For example, C2 fromthe N-terminus of the polypeptide of SEQ ID NO:2 is the cysteine atamino acid position 48, or position 49 (additional N-terminal Met) ifexpressed in E. coli (see, for example, SEQ ID NO:13). This secondcysteine (of which there are seven, like IL-28B) or C2 of IL-28A can bemutated, for example, to a serine, alanine, threonine, valine, orasparagine. IL-28A C2 mutant molecules of the present invention include,for example, polynucleotide molecules as shown in SEQ ID NOs:18 and 20,including DNA and RNA molecules, that encode IL-28A C2 mutantpolypeptides as shown in SEQ ID NOs:19 and 21, respectively. AdditionalIL-28A C2 mutant molecules of the present invention include polypeptidesas shown in SEQ ID NOs:36, 37 and 163.

The present invention also includes biologically active mutants ofIL-28A C2 cysteine mutants which provide, at least partially, anti-tumoractivity and/or immune regulatory activity. The second cysteine or C2from the N-terminus of IL-28A can mutated to any amino acid that doesnot form a disulfide bond with another cysteine, e.g., serine, alanine,threonine, valine or aspargine. The biologically active mutants ofIL-28A C2 cysteine mutants of the present invention include N-, C-, andN- and C-terminal deletions of IL-28A, e.g., the polypeptide of SEQ IDNO:163 encoded by the polynucleotide of SEQ ID NO:162.

N-terminally modified biologically active mutants of IL-28A C2 mutantsinclude, for example, amino acid residues 3-176 of SEQ ID NO:163 whichis encoded by nucleotides 7-528 of SEQ ID NO:162; amino acid residues4-176 of SEQ ID NO:163 which is encoded by nucleotides 10-528 of SEQ IDNO:162; amino acid residues 5-176 of SEQ ID NO:163 which is encoded bynucleotides 13-528 of SEQ ID NO:162; amino acid residues 6-176 of SEQ IDNO:163 which is encoded by nucleotides 16-528 of SEQ ID NO:162; aminoacid residues 7-176 of SEQ ID NO:163 which is encoded by nucleotidies19-528 of SEQ ID NO:162; amino acid residues 8-176 of SEQ ID NO:163which is encoded by nucleotides 22-528 of SEQ ID NO:162; amino acidresidues 9-176 of SEQ ID NO:163 which is encoded by nucleotides 25-528of SEQ ID NO:162; amino acid residues 10-176 of SEQ ID NO:163 which isencoded by nucleotides 28-528 of SEQ ID NO:162; amino acid residues11-176 of SEQ ID NO:163 which is encoded by nucleotides 31-528 of SEQ IDNO:162; amino acid residues 12-176 of SEQ ID NO:163 which is encoded bynucleotides 34-528 of SEQ ID NO:162; amino acid residues 13-176 of SEQID NO:163 which is encoded by nucleotides 37-528 of SEQ ID NO:162; aminoacid residues 14-176 of SEQ ID NO:163 which is encoded by nucleotides40-528 of SEQ ID NO:162; amino acid residues 15-176 of SEQ ID NO:163which is encoded by nucleotides 43-528 of SEQ ID NO:162; and amino acidresidues 16-176 of SEQ ID NO:163 which is encoded by nucleotides 46-528of SEQ ID NO:162. The N-terminally modified biologically active mutantsof IL-28A C2 mutants of the present invention may also include anN-terminal Methione if expressed, for instance, in E. coli.

C-terminally modified biologically active mutants of IL-28A C2 mutantsinclude, for example, amino acid residues 1-175 of SEQ ID NO:163 whichis encoded by nucleotides 1-525 of SEQ ID NO:162.

N-terminally and C-terminally modified biologically active mutants ofIL-28A C2 mutants include, for example, amino acid residues 2-175 of SEQID NO:163 which is encoded by nucleotides 4-525 of SEQ ID NO:162; aminoacid residues 3-175 of SEQ ID NO:163 which is encoded by nucleotides7-525 of SEQ ID NO:162; amino acid residues 4-175 of SEQ ID NO:163 whichis encoded by nucleotides 10-525 of SEQ ID NO:162; amino acid residues5-175 of SEQ ID NO:163 which is encoded by nucleotides 13-525 of SEQ IDNO:162; amino acid residues 6-175 of SEQ ID NO:163 which is encoded bynucleotides 16-525 of SEQ ID NO:162; amino acid residues 7-175 of SEQ IDNO:163 which is encoded by nucleotides 19-525 of SEQ ID NO:162; aminoacid residues 8-175 of SEQ ID NO:163 which is encoded by nucleotides22-525 of SEQ ID NO:162; amino acid residues 9-175 of SEQ ID NO:163which is encoded by nucleotides 25-525 of SEQ ID NO:162; amino acidresidues 10-175 of SEQ ID NO:163 which is encoded by nucleotides 28-525of SEQ ID NO:162; amino acid residues 11-175 of SEQ ID NO:163 which isencoded by nucleotides 31-525 of SEQ ID NO:162; amino acid residues12-175 of SEQ ID NO:163 which is encoded by nucleotides 34-525 of SEQ IDNO:162; amino acid residues 13-175 of SEQ ID NO:163 which is encoded bynucleotides 37-525 of SEQ ID NO:162; amino acid residues 14-175 of SEQID NO:163 which is encoded by nucleotides 40-525 of SEQ ID NO:162; aminoacid residues 15-175 of SEQ ID NO:163 which is encoded by nucleotides43-525 of SEQ ID NO:162; amino acid residues 16-175 of SEQ ID NO:163which is encoded by nucleotides 46-525 of SEQ ID NO:162; and amino acidresidues 17-175 of SEQ ID NO:163 which is encoded by nucleotides 49-525of SEQ ID NO:162. The N-terminally and C-terminally modifiedbiologically active mutants of IL-28A C2 mutants of the presentinvention may also include an N-terminal Methione if expressed, forinstance, in E. coli.

In addition to the IL-28A C2 mutants, the present invention alsoincludes IL-28A polypeptides comprising a mutation at the third cysteineposition, C3, of the mature polypeptide. For example, C3 from theN-terminus of the polypeptide of SEQ ID NO:2, is the cysteine atposition 50, or position 51 (additional N-terminal Met) if expressed inE. coli (see, for example, SEQ ID NO:13). IL-28A C3 mutant molecules ofthe present invention include, for example, polynucleotide molecules asshown in SEQ ID NOs:22 and 24, including DNA and RNA molecules, thatencode IL-28A C3 mutant polypeptides as shown in SEQ ID NOs:23 and 25,respectively. Additional IL-28A C3 mutant molecules of the presentinvention include polypeptides as shown in SEQ ID NOs:38, 39 and 165.

The present invention also includes biologically active mutants ofIL-28A C3 cysteine mutants which provide, at least partially, anti-tumoractivity and/or immune regulatory activity. The third cysteine or C3from the N-terminus of IL-28A can mutated to any amino acid that doesnot form a disulfide bond with another cysteine, e.g., serine, alanine,threonine, valine or aspargine. The biologically active mutants ofIL-28A C3 cysteine mutants of the present invention include N-, C-, andN- and C-terminal deletions of IL-28A, e.g., the polypeptide of SEQ IDNO:165 encoded by the polynucleotide of SEQ ID NO:164.

N-terminally modified biologically active mutants of IL-28A C3 mutantsinclude, for example, amino acid residues 2-176 of SEQ ID NO:165 whichis encoded by nucleotides 4-528 of SEQ ID NO:164; amino acid residues3-176 of SEQ ID NO:165 which is encoded by nucleotides 7-528 of SEQ IDNO:164; amino acid residues 4-176 of SEQ ID NO:165 which is encoded bynucleotides 10-528 of SEQ ID NO:164; amino acid residues 5-176 of SEQ IDNO:165 which is encoded by nucleotides 13-528 of SEQ ID NO:164; aminoacid residues 6-176 of SEQ ID NO:165 which is encoded by nucleotides16-528 of SEQ ID NO:164; amino acid residues 7-176 of SEQ ID NO:165which is encoded by nucleotidies 19-528 of SEQ ID NO:164; amino acidresidues 8-176 of SEQ ID NO:165 which is encoded by nucleotides 22-528of SEQ ID NO:164; amino acid residues 9-176 of SEQ ID NO:165 which isencoded by nucleotides 25-528 of SEQ ID NO:164; amino acid residues10-176 of SEQ ID NO:165 which is encoded by nucleotides 28-528 of SEQ IDNO:164; amino acid residues 11-176 of SEQ ID NO:165 which is encoded bynucleotides 31-528 of SEQ ID NO:164; amino acid residues 12-176 of SEQID NO:165 which is encoded by nucleotides 34-528 of SEQ ID NO:164; aminoacid residues 13-176 of SEQ ID NO:165 which is encoded by nucleotides37-528 of SEQ ID NO:164; amino acid residues 14-176 of SEQ ID NO:165which is encoded by nucleotides 40-528 of SEQ ID NO:164; amino acidresidues 15-176 of SEQ ID NO:165 which is encoded by nucleotides 43-528of SEQ ID NO:164; and amino acid residues 16-176 of SEQ ID NO:165 whichis encoded by nucleotides 46-528 of SEQ ID NO:164. The N-terminallymodified biologically active mutants of IL-28A C3 mutants of the presentinvention may also include an N-terminal Methione if expressed, forinstance, in E. coli.

C-terminally modified biologically active mutants of IL-28A C3 mutantsinclude, for example, amino acid residues 1-175 of SEQ ID NO:165 whichis encoded by nucleotides 1-525 of SEQ ID NO:164.

N-terminally and C-terminally modified biologically active mutants ofIL-28A C3 mutants include, for example, amino acid residues 2-175 of SEQID NO:165 which is encoded by nucleotides 4-525 of SEQ ID NO:164; aminoacid residues 3-175 of SEQ ID NO:165 which is encoded by nucleotides7-525 of SEQ ID NO:164; amino acid residues 4-175 which is encoded bynucleotides 10-525 of SEQ ID NO:164; amino acid residues 5-175 of SEQ IDNO:165 which is encoded by nucleotides 13-525 of SEQ ID NO:164; aminoacid residues 6-175 of SEQ ID NO:165 which is encoded by nucleotides16-525 of SEQ ID NO:164; amino acid residues 7-175 of SEQ ID NO:165which is encoded by nucleotides 19-525 of SEQ ID NO:164; amino acidresidues 8-175 of SEQ ID NO:165 which is encoded by nucleotides 22-525of SEQ ID NO:164; amino acid residues 9-175 of SEQ ID NO:165 which isencoded by nucleotides 25-525 of SEQ ID NO:164; amino acid residues10-175 of SEQ ID NO:165 which is encoded by nucleotides 28-525 of SEQ IDNO:164; amino acid residues 11-175 of SEQ ID NO:165 which is encoded bynucleotides 31-525 of SEQ ID NO:164; amino acid residues 12-175 of SEQID NO:165 which is encoded by nucleotides 34-525 of SEQ ID NO:164; aminoacid residues 13-175 of SEQ ID NO:165 which is encoded by nucleotides37-525 of SEQ ID NO:164; amino acid residues 14-175 of SEQ ID NO:165which is encoded by nucleotides 40-525 of SEQ ID NO:164; amino acidresidues 15-175 of SEQ ID NO:165 which is encoded by nucleotides 43-525of SEQ ID NO:164; amino acid residues 16-175 of SEQ ID NO:165 which isencoded by nucleotides 46-525 of SEQ ID NO:164; and amino acid residues17-175 of SEQ ID NO:165 which is encoded by nucleotides 49-525 of SEQ IDNO:164. The N-terminally and C-terminally modified biologically activemutants of IL-28A C3 mutants of the present invention may also includean N-terminal Methione if expressed, for instance, in E. coli.

The IL-28A polypeptides of the present invention include, for example,SEQ ID NOs:2, 13, 19, 21, 23, 25, 163 and 165 which are encoded byIL-28A polynucleotide molecules as shown in SEQ ID NOs:1, 12, 18, 20,22, 24, 162 and 164, respectively. In addition, the present inventionalso provides for IL-28A polypeptides as shown in SEQ ID NOs:36, 37, 38,and 39, C2 mutants thereof, N-terminally modified C2 mutants thereof,C-terminally modified C2 mutants thereof, N-terminally and C-terminallyC2 mutants thereof, C3 mutants thereof, N-terminally modified C3 mutantsthereof, C-terminally modified C3 mutants thereof, N-terminally andC-terminally modified C3 mutants thereof, fragments thereof, and fusionproteins thereof.

A Zcyto22 or IL-28B gene encodes a polypeptide of 205 amino acids, asshown in SEQ ID NO:6. The signal sequence for IL-28B comprises aminoacid residue-25 (Met) through amino acid residue 0 (Ala) of SEQ ID NO:6,or alternatively amino acid residues-21 (Met) through amino acid residue0 (Ala) of SEQ ID NO:6. The mature peptide for IL-28B begins at aminoacid residue 1 (Val) of SEQ ID NO:6. IL-28B helices are predicted asfollow: helix A is defined by amino acid residues 8 (Leu) to 41 (Glu);helix B by amino acid residues 58 (Trp) to 65 (Gln); helix C by aminoacid residues 69 (Arg) to 86 (Ala); helix D by amino acid residues 95(Gly) to 114 (Ala); helix E by amino acid residues 126 (Thr) to 142(Lys); and helix F by amino acid residues 148 (Cys) to 169 (Ala); asshown in SEQ ID NO:6. When a polynucleotide sequence encoding the maturepolypeptide is expressed in a prokaryotic system, such as E. coli, the asecretory signal sequence may not be required and the an N-terminal Metwill be present, resulting in expression of a polypeptide such as isshown in SEQ ID NO:17.

IL-28B polypeptides of the present invention also include a mutation atthe second cysteine, C2, of the mature polypeptide. For example, C2 fromthe N-terminus of the polypeptide of SEQ ID NO:6 is the cysteine atamino acid position 48, or position 49 (additional N-terminal Met) ifexpressed in E. coli (see, for example, SEQ ID NO:17). This secondcysteine (of which there are seven, like IL-28A) or C2 of IL-28B can bemutated, for example, to a serine, alanine, threonine, valine, orasparagine. IL-28B C2 mutant molecules of the present invention include,for example, polynucleotide molecules as shown in SEQ ID NOs:122 and124, including DNA and RNA molecules, that encode IL-28B C2 mutantpolypeptides as shown in SEQ ID NOs:123 and 125, respectively.Additional IL-28B C2 mutant molecules of the present invention includepolynucleotide molecules as shown in SEQ ID NOs:130 and 132 includingDNA and RNA molecules, that encode IL-28B C2 mutant polypeptides asshown in SEQ ID NOs:131 and 133, respectively (PCT publication WO03/066002 (Kotenko et al.)).

The present invention also includes biologically active mutants ofIL-28B C2 cysteine mutants which provide, at least partially, anti-tumoractivity and/or immune regulatory activity. The second cysteine or C2from the N-terminus of IL-28B can mutated to any amino acid that doesnot form a disulfide bond with another cysteine, e.g., serine, alanine,threonine, valine or aspargine. The biologically active mutants ofIL-28B C2 cysteine mutants of the present invention include N-, C-, andN- and C-terminal deletions of IL-28B, e.g., the polypeptide of SEQ IDNO:167 encoded by the polynucleotide of SEQ ID NO:166.

N-terminally modified biologically active mutants of IL-28B C2 mutantsinclude, for example, amino acid residues 2-176 of SEQ ID NO:167 whichis encoded by nucleotides 4-528 of SEQ ID NO:166; amino acid residues3-176 of SEQ ID NO:167 which is encoded by nucleotides 7-528 of SEQ IDNO:166; amino acid residues 4-176 of SEQ ID NO:167 which is encoded bynucleotides 10-528 of SEQ ID NO:166; amino acid residues 5-176 of SEQ IDNO:167 which is encoded by nucleotides 13-528 of SEQ ID NO:166; aminoacid residues 6-176 of SEQ ID NO:167 which is encoded by nucleotides16-528 of SEQ ID NO:166; amino acid residues 7-176 of SEQ ID NO:167which is encoded by nucleotides 19-528 of SEQ ID NO:166; amino acidresidues 8-176 of SEQ ID NO:167 which is encoded by nucleotides 22-528of SEQ ID NO:166; amino acid residues 9-176 of SEQ ID NO:167 which isencoded by nucleotides 25-528 of SEQ ID NO:166; amino acid residues10-176 of SEQ ID NO:167 which is encoded by nucleotides 28-528 of SEQ IDNO:166; amino acid residues 11-176 of SEQ ID NO:167 which is encoded bynucleotides 31-528 of SEQ ID NO:166; amino acid residues 12-176 of SEQID NO:167 which is encoded by nucleotides 34-528 of SEQ ID NO:166; aminoacid residues 13-176 of SEQ ID NO:167 which is encoded by nucleotides37-528 of SEQ ID NO:166; amino acid residues 14-176 of SEQ ID NO:167which is encoded by nucleotides 40-528 of SEQ ID NO:166; amino acidresidues 15-176 of SEQ ID NO:167 which is encoded by nucleotides 43-528of SEQ ID NO:166; amino acid residues 16-176 of SEQ ID NO:167 which isencoded by nucleotides 46-528 of SEQ ID NO:166; and amino acid residues17-176 of SEQ ID NO:167 which is encoded by nucleotides 49-528 of SEQ IDNO:166. The N-terminally modified biologically active mutants of IL-28C2 mutants of the present invention may also include an N-terminalMethione if expressed, for instance, in E. coli.

C-terminally modified biologically active mutants of IL-28B C2 mutantsinclude, for example, amino acid residues 1-175 of SEQ ID NO:167 whichis encoded by nucleotides 1-525 of SEQ ID NO:166.

N-terminally and C-terminally biologically active mutants of IL-28B C2mutants include, for example, amino acid residues 2-175 of SEQ ID NO:167which is encoded by nucleotides 4-525 of SEQ ID NO:166; amino acidresidues 3-175 of SEQ ID NO:167 which is encoded by nucleotides 7-525 ofSEQ ID NO:166; amino acid residues 4-175 of SEQ ID NO:167 which isencoded by nucleotides 10-525 of SEQ ID NO:166; amino acid residues5-175 of SEQ ID NO:167 which is encoded by nucleotides 13-525 of SEQ IDNO:166; amino acid residues 6-175 of SEQ ID NO:167 which is encoded bynucleotides 16-525 of SEQ ID NO:166; amino acid residues 7-175 of SEQ IDNO:167 which is encoded by nucleotides 19-525 of SEQ ID NO:166; aminoacid residues 8-175 of SEQ ID NO:167 which is encoded by nucleotides22-525 of SEQ ID NO:166; amino acid residues 9-175 of SEQ ID NO:167which is encoded by nucleotides 25-525 of SEQ ID NO:166; amino acidresidues 10-175 of SEQ ID NO:167 which is encoded by nucleotides 28-525of SEQ ID NO:166; amino acid residues 11-175 of SEQ ID NO:167 which isencoded by nucleotides 31-525 of SEQ ID NO:166; amino acid residues12-175 of SEQ ID NO:167 which is encoded by nucleotides 34-525 of SEQ IDNO:166; amino acid residues 13-175 of SEQ ID NO:167 which is encoded bynucleotides 37-525 of SEQ ID NO:166; amino acid residues 14-175 of SEQID NO:167 which is encoded by nucleotides 40-525 of SEQ ID NO:166; aminoacid residues 15-175 of SEQ ID NO:167 which is encoded by nucleotides43-525 of SEQ ID NO:166; amino acid residues 16-175 of SEQ ID NO:167which is encoded by nucleotides 46-525 of SEQ ID NO:166; and amino acidresidues 17-175 of SEQ ID NO:167 which is encoded by nucleotides 49-525of SEQ ID NO:166. The N-terminally and C-terminally modifiedbiologically active mutants of IL-28 C2 mutants of the present inventionmay also include an N-terminal Methione if expressed, for instance, inE. coli.

In addition to the IL-28B C2 mutants, the present invention alsoincludes IL-28B polypeptides comprising a mutation at the third cysteineposition, C3, of the mature polypeptide. For example, C3 from theN-terminus of the polypeptide of SEQ ID NO:6, is the cysteine atposition 50, or position 51 (additional N-terminal Met) if expressed inE. coli (see, for example, SEQ ID NO:17). IL-28B C3 mutant molecules ofthe present invention include, for example, polynucleotide molecules asshown in SEQ ID NOs:126 and 128, including DNA and RNA molecules, thatencode IL-28B C3 mutant polypeptides as shown in SEQ ID NOs:127 and 129,respectively (PCT publication WO 03/066002 (Kotenko et al.)). AdditionalIL-28B C3 mutant molecules of the present invention includepolynucleotide molecules as shown in SEQ ID NOs:134 and 136 includingDNA and RNA molecules, that encode IL-28B C3 mutant polypeptides asshown in SEQ ID NOs:135 and 137, respectively (PCT publication WO03/066002 (Kotenko et al.)).

N-terminally biologically active mutants of IL-28B C3 mutants include,for example, amino acid residues 2-176 of SEQ ID NO:169 which is encodedby nucleotides 4-528 of SEQ ID NO:168; amino acid residues 3-176 of SEQID NO:169 which is encoded by nucleotides 7-528 of SEQ ID NO:168; aminoacid residues 4-176 of SEQ ID NO:169 which is encoded by nucleotides10-528 of SEQ ID NO:168; amino acid residues 5-176 of SEQ ID NO:169which is encoded by nucleotides 13-528 of SEQ ID NO:168; amino acidresidues 6-176 of SEQ ID NO:169 which is encoded by nucleotides 16-528of SEQ ID NO:168; amino acid residues 7-176 of SEQ ID NO:169 which isencoded by nucleotides 19-528 of SEQ ID NO:168; amino acid residues8-176 of SEQ ID NO:169 which is encoded by nucleotides 22-528 of SEQ IDNO:168; amino acid residues 9-176 of SEQ ID NO:169 which is encoded bynucleotides 25-528 of SEQ ID NO:168; amino acid residues 10-176 of SEQID NO:169 which is encoded by nucleotides 28-528 of SEQ ID NO:168; aminoacid residues 11-176 of SEQ ID NO:169 which is encoded by nucleotides31-528 of SEQ ID NO:168; amino acid residues 12-176 of SEQ ID NO:169which is encoded by nucleotides 34-528 of SEQ ID NO:168; amino acidresidues 13-176 of SEQ ID NO:169 which is encoded by nucleotides 37-528of SEQ ID NO:168; amino acid residues 14-176 of SEQ ID NO:169 which isencoded by nucleotides 40-528 of SEQ ID NO:168; amino acid residues15-176 of SEQ ID NO:169 which is encoded by nucleotides 43-528 of SEQ IDNO:168; amino acid residues 16-176 of SEQ ID NO:169 which is encoded bynucleotides 46-528 of SEQ ID NO:168; and amino acid residues 17-176 ofSEQ ID NO:169 which is encoded by nucleotides 49-528 of SEQ ID NO:168.The N-terminally modified biologically active mutants of IL-28 C3mutants of the present invention may also include an N-terminal Methioneif expressed, for instance, in E. coli.

C-terminally modified biologically active mutants of IL-28B C3 mutantsinclude, for example, amino acid residues 1-175 of SEQ ID NO:169 whichis encoded by nucleotides 1-525 of SEQ ID NO:168.

N-terminally and C-terminally biologically active mutants of IL-28B C3mutants include, for example, amino acid residues 2-175 of SEQ ID NO:169which is encoded by nucleotides 4-525 of SEQ ID NO:168; amino acidresidues 3-175 of SEQ ID NO:169 which is encoded by nucleotides 7-525 ofSEQ ID NO:168; amino acid residues 4-175 of SEQ ID NO:169 which isencoded by nucleotides 10-525 of SEQ ID NO:168; amino acid residues5-175 of SEQ ID NO:169 which is encoded by nucleotides 13-525 of SEQ IDNO:168; amino acid residues 6-175 of SEQ ID NO:169 which is encoded bynucleotides 16-525 of SEQ ID NO:168; amino acid residues 7-175 of SEQ IDNO:169 which is encoded by nucleotides 19-525 of SEQ ID NO:168; aminoacid residues 8-175 of SEQ ID NO:169 which is encoded by nucleotides22-525 of SEQ ID NO:168; amino acid residues 9-175 of SEQ ID NO:169which is encoded by nucleotides 25-525 of SEQ ID NO:168; amino acidresidues 10-175 of SEQ ID NO:169 which is encoded by nucleotides 28-525of SEQ ID NO:168; amino acid residues 11-175 of SEQ ID NO:169 which isencoded by nucleotides 31-525 of SEQ ID NO:168; amino acid residues12-175 of SEQ ID NO:169 which is encoded by nucleotides 34-525 of SEQ IDNO:168; amino acid residues 13-175 of SEQ ID NO:169 which is encoded bynucleotides 37-525 of SEQ ID NO:168; amino acid residues 14-175 of SEQID NO:169 which is encoded by nucleotides 40-525 of SEQ ID NO:168; aminoacid residues 15-175 of SEQ ID NO:169 which is encoded by nucleotides43-525 of SEQ ID NO:168; amino acid residues 16-175 of SEQ ID NO:169which is encoded by nucleotides 46-525 of SEQ ID NO:168; and amino acidresidues 17-175 of SEQ ID NO:169 which is encoded by nucleotides 49-525of SEQ ID NO:168. The N-terminally and C-terminally modifiedbiologically active mutants of IL-28 C3 mutants of the present inventionmay also include an N-terminal Methione if expressed, for instance, inE. coli.

The IL-28B polypeptides of the present invention include, for example,SEQ ID NOs:6, 17, 123, 125, 127, 129, 131, 133, 135, 137, 167 and 169,which are encoded by IL-28B polynucleotide molecules as shown in SEQ IDNOs:5, 16, 122, 124, 126, 128, 130, 132, 134, 136, 166 and 168,respectively, C2 mutants thereof, N-terminally modified C2 mutantsthereof, C-terminally modified C2 mutants thereof, N-terminally andC-terminally C2 mutants thereof, C3 mutants thereof, N-terminallymodified C3 mutants thereof, C-terminally modified C3 mutants thereof,N-terminally and C-terminally modified C3 mutants thereof, fragmentsthereof, and fusion proteins thereof.

Zcyto21 or IL-29 polypeptides of the present invention also include amutation at the fifth cysteine, C5, of the mature polypeptide. Forexample, C5 from the N-terminus of the polypeptide of SEQ ID NO:4, isthe cysteine at position 171, or position 172 (additional N-terminalMet) if expressed in E. coli. (see, for example, SEQ ID NO:15). Thisfifth cysteine or C5 of IL-29 can be mutated, for example, to a serine,alanine, threonine, valine, or asparagine. These IL-29 C5 mutantpolypeptides have a disulfide bond pattern of C1(Cys15 of SEQ IDNO:4)/C3(Cys112 of SEQ ID NO:4) and C2(Cys49 of SEQ ID NO:4)/C4(Cys145of SEQ ID NO:4). Additional IL-29 C5 mutant molecules of the presentinvention include polynucleotide molecules as shown in SEQ ID NOs:26,28, 82, 84, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, and160, including DNA and RNA molecules, that encode IL-29 C5 mutantpolypeptides as shown in SEQ ID NOs:27, 29, 83, 85, 139, 141, 143, 145,147, 149, 151, 153, 155, 157, 159, and 161, respectively. AdditionalIL-29 C5 mutant molecules of the present invention includepolynucleotide molecules as shown in SEQ ID NOs:86, 88, 94, and 96,including DNA and RNA molecules, that encode IL-29 C5 mutantpolypeptides as shown in SEQ ID NOs:87, 89, 95, and 97, respectively(PCT publication WO 03/066002 (Kotenko et al.)). Additional, IL-29 C5mutant molecules of the present invention include polynucleotidemolecules as shown in SEQ ID NOs:102, 104, 110, and 112, including DNAand RNA molecules, that encode IL-29 C5 mutant polypeptides as shown inSEQ ID NOs:103, 105, 111, and 113, respectively (PCT publication WO02/092762 (Baum et al.)).

The present invention also includes biologically active mutants of IL-29C5 cysteine mutants which provide, at least partially, anti-tumoractivity and/or immune regulatory activity. The fifth cysteine or C5from the N-terminus of IL-29 can mutated to any amino acid that does notform a disulfide bond with another cysteine, e.g., serine, alanine,threonine, valine or aspargine. The biologically active mutants of IL-29C5 cysteine mutants of the present invention include N-, C-, and N- andC-terminal deletions of IL-29, e.g., the polypeptides of SEQ ID NOs:173and 175 encoded by the polynucleotides of SEQ ID NOs:172 and 174,respectively.

N-terminally modified biologically active mutants of IL-29 C5 mutantsinclude, for example, amino acid residues 2-182 of SEQ ID NO:173 whichis encoded by nucleotides 4-546 of SEQ ID NO:172; amino acid residues3-182 of SEQ ID NO:173 which is encoded by nucleotides 7-546 of SEQ IDNO:172; amino acid residues 4-182 of SEQ ID NO:173 which is encoded bynucleotides 10-546 of SEQ ID NO:172; amino acid residues 5-182 of SEQ IDNO:173 which is encoded by nucleotides 13-546 of SEQ ID NO:172; aminoacid residues 6-182 of SEQ ID NO:173 which is encoded by nucleotides16-546 of SEQ ID NO:172; amino acid residues 7-182 of SEQ ID NO:173which is encoded by nucleotides 19-546 of SEQ ID NO:172; amino acidresidues 8-182 of SEQ ID NO:173 which is encoded by nucleotides 22-546of SEQ ID NO:172; amino acid residues 9-182 of SEQ ID NO:173 which isencoded by nucleotides 25-546 of SEQ ID NO:172; amino acid residues10-182 of SEQ ID NO:173 which is encoded by nucleotides 28-546 of SEQ IDNO:172; amino acid residues 11-182 of SEQ ID NO:173 which is encoded bynucleotides 31-546 of SEQ ID NO:172; amino acid residues 12-182 of SEQID NO:173 which is encoded by nucleotides 34-546 of SEQ ID NO:172; aminoacid residues 13-182 of SEQ ID NO:173 which is encoded by nucleotides37-546 of SEQ ID NO:172; amino acid residues 14-182 of SEQ ID NO:173which is encoded by nucleotides 40-546 of SEQ ID NO:172; amino acidresidues 15-182 of SEQ ID NO:173 which is encoded by nucleotides 43-546of SEQ ID NO:172; amino acid residues 2-176 of SEQ ID NO:150 which isencoded by nucleotides 4-528 of SEQ ID NO:149; amino acid residues 3-176of SEQ ID NO:150 which is encoded by nucleotides 7-528 of SEQ ID NO:149;amino acid residues 4-176 of SEQ ID NO:150 which is encoded bynucleotides 10-528 of SEQ ID NO:149; amino acid residues 5-176 of SEQ IDNO:150 which is encoded by nucleotides 13-528 of SEQ ID NO:149; aminoacid residues 6-176 of SEQ ID NO:150 which is encoded by nucleotides16-528 of SEQ ID NO:149; amino acid residues 7-176 of SEQ ID NO:150which is encoded by nucleotides 19-528 of SEQ ID NO:149; amino acidresidues 8-176 of SEQ ID NO:150 which is encoded by nucleotides 22-528of SEQ ID NO:149; and amino acid residues 9-176 of SEQ ID NO:150 whichis encoded by nucleotides 25-528 of SEQ ID NO:149. The N-terminallymodified biologically active mutants of IL-29 C5 mutants of the presentinvention may also include an N-terminal Methione if expressed, forinstance, in E. coli.

C-terminally modified biologically active mutants of IL-29 C5 mutantsinclude, for example, amino acid residues 1-181 of SEQ ID NO:173 whichis encoded by nucleotides 1-543 of SEQ ID NO:172; amino acid residues1-180 of SEQ ID NO:173 which is encoded by nucleotides 1-540 of SEQ IDNO:172; amino acid residues 1-179 of SEQ ID NO:173 which is encoded bynucleotides 1-537 of SEQ ID NO:172; amino acid residues 1-178 of SEQ IDNO:173 which is encoded by nucleotides 1-534 of SEQ ID NO:172; aminoacid residues 1-177 of SEQ ID NO:173 which is encoded by nucleotides1-531 of SEQ ID NO:172; amino acid residues 1-176 of SEQ ID NO:173 whichis encoded by nucleotides 1-528 of SEQ ID NO:172; amino acid residues1-175 of SEQ ID NO:173 which is encoded by nucleotides 1-525 of SEQ IDNO:172; amino acid residues 1-174 of SEQ ID NO:173 which is encoded bynucleotides 1-522 of SEQ ID NO:172; amino acid residues 1-173 of SEQ IDNO:173 which is encoded by nucleotides 1-519 of SEQ ID NO:172; aminoacid residues 1-172 of SEQ ID NO:173 which is encoded by nucleotides1-516 of SEQ ID NO:172; amino acid residues 1-175 of SEQ ID NO:150 whichis encoded by nucleotides 1-525 of SEQ ID NO:149; amino acid residues1-174 of SEQ ID NO:150 which is encoded by nucleotides 1-522 of SEQ IDNO:149; amino acid residues 1-173 of SEQ ID NO:150 which is encoded bynucleotides 1-519 of SEQ ID NO:149; amino acid residues 1-172 of SEQ IDNO:150 which is encoded by nucleotides 1-516 of SEQ ID NO:149; aminoacid residues 1-171 of SEQ ID NO:150 which is encoded by nucleotides1-513 of SEQ ID NO:149; amino acid residues 1-170 of SEQ ID NO:150 whichis encoded by nucleotides 1-510 of SEQ ID NO:149; amino acid residues1-169 of SEQ ID NO:150 which is encoded by nucleotides 1-507 of SEQ IDNO:149; amino acid residues 1-168 of SEQ ID NO:150 which is encoded bynucleotides 1-504 of SEQ ID NO:149; amino acid residues 1-167 of SEQ IDNO:150 which is encoded by nucleotides 1-501 of SEQ ID NO:149; and aminoacid residues 1-166 of SEQ ID NO:150 which is encoded by nucleotides1-498 of SEQ ID NO:149.

N-terminally and C-terminally modified biologically active mutants ofIL-29 C5 mutants include, for example, amino acid residues 2-182 of SEQID NO:173 which is encoded by nucleotides 4-546 of SEQ ID NO:172; aminoacid residues 2-181 of SEQ ID NO:173 which is encoded by nucleotides4-543 of SEQ ID NO:172; amino acid residues 2-180 of SEQ ID NO:173 whichis encoded by nucleotides 4-540 of SEQ ID NO:172; amino acid residues2-179 of SEQ ID NO:173 which is encoded by nucleotides 4-537 of SEQ IDNO:172; amino acid residues 2-178 of SEQ ID NO:173 which is encoded bynucleotides 4-534 of SEQ ID NO:172; amino acid residues 2-177 of SEQ IDNO:173 which is encoded by nucleotides 4-531 of SEQ ID NO:172; aminoacid residues 2-176 of SEQ ID NO:173 which is encoded by nucleotides4-528 of SEQ ID NO:172; amino acid residues 2-175 of SEQ ID NO:173 whichis encoded by nucleotides 4-525 of SEQ ID NO:172; amino acid residues2-174 of SEQ ID NO:173 which is encoded by nucleotides 4-522 of SEQ IDNO:172; amino acid residues 2-173 of SEQ ID NO:173 which is encoded bynucleotides 4-519 of SEQ ID NO:172; amino acid residues 2-172 of SEQ IDNO:173 which is encoded by nucleotides 4-516 of SEQ ID NO:172; aminoacid residues 3-182 of SEQ ID NO:173 which is encoded by nucleotides7-546 of SEQ ID NO:172; amino acid residues 3-181 of SEQ ID NO:173 whichis encoded by nucleotides 7-543 of SEQ ID NO:172; amino acid residues3-180 of SEQ ID NO:173 which is encoded by nucleotides 7-540 of SEQ IDNO:172; amino acid residues 3-179 of SEQ ID NO:173 which is encoded bynucleotides 7-537 of SEQ ID NO:172; amino acid residues 3-178 of SEQ IDNO:173 which is encoded by nucleotides 7-534 of SEQ ID NO:172; aminoacid residues 3-177 of SEQ ID NO:173 which is encoded by nucleotides7-531 of SEQ ID NO:172; amino acid residues 3-176 of SEQ ID NO:173 whichis encoded by nucleotides 7-528 of SEQ ID NO:172; amino acid residues3-175 of SEQ ID NO:173 which is encoded by nucleotides 7-525 of SEQ IDNO:172; amino acid residues 3-174 of SEQ ID NO:173 which is encoded bynucleotides 7-522 of SEQ ID NO:172; amino acid residues 3-173 of SEQ IDNO:173 which is encoded by nucleotides 7-519 of SEQ ID NO:172; aminoacid residues 3-172 of SEQ ID NO:173 which is encoded by nucleotides7-516 of SEQ ID NO:172; amino acid residues 4-182 of SEQ ID NO:173 whichis encoded by nucleotides 10-546 of SEQ ID NO:172; amino acid residues4-181 of SEQ ID NO:173 which is encoded by nucleotides 10-543 of SEQ IDNO:172; amino acid residues 4-180 of SEQ ID NO:173 which is encoded bynucleotides 10-540 of SEQ ID NO:172; amino acid residues 4-179 of SEQ IDNO:173 which is encoded by nucleotides 10-537 of SEQ ID NO:172; aminoacid residues 4-178 of SEQ ID NO:173 which is encoded by nucleotides10-534 of SEQ ID NO:147; amino acid residues 4-177 of SEQ ID NO:173which is encoded by nucleotides 10-531 of SEQ ID NO:147; amino acidresidues 4-176 of SEQ ID NO:173 which is encoded by nucleotides 10-528of SEQ ID NO:147; amino acid residues 4-175 of SEQ ID NO:173 which isencoded by nucleotides 10-525 of SEQ ID NO:147; amino acid residues4-174 of SEQ ID NO:173 which is encoded by nucleotides 10-522 of SEQ IDNO:147; amino acid residues 4-173 of SEQ ID NO:173 which is encoded bynucleotides 10-519 of SEQ ID NO:172; amino acid residues 4-172 of SEQ IDNO:173 which is encoded by nucleotides 10-516 of SEQ ID NO:172; aminoacid residues 5-182 of SEQ ID NO:173 which is encoded by nucleotides13-546 of SEQ ID NO:172; amino acid residues 5-181 of SEQ ID NO:173which is encoded by nucleotides 13-543 of SEQ ID NO:172; amino acidresidues 5-180 of SEQ ID NO:173 which is encoded by nucleotides 13-540of SEQ ID NO:172; amino acid residues 5-179 of SEQ ID NO:173 which isencoded by nucleotides 13-537 of SEQ ID NO:172; amino acid residues5-178 of SEQ ID NO:173 which is encoded by nucleotides 13-534 of SEQ IDNO:172; amino acid residues 5-177 of SEQ ID NO:173 which is encoded bynucleotides 13-531 of SEQ ID NO:172; amino acid residues 5-176 of SEQ IDNO:173 which is encoded by nucleotides 13-528 of SEQ ID NO:172; aminoacid residues 5-175 of SEQ ID NO:173 which is encoded by nucleotides13-525 of SEQ ID NO:172; amino acid residues 5-174 of SEQ ID NO:173which is encoded by nucleotides 13-522 of SEQ ID NO:172; amino acidresidues 5-173 of SEQ ID NO:173 which is encoded by nucleotides 13-519of SEQ ID NO:172; amino acid residues 5-172 of SEQ ID NO:173 which isencoded by nucleotides 13-516 of SEQ ID NO:172; amino acid residues6-182 of SEQ ID NO:173 which is encoded by nucleotides 16-546 of SEQ IDNO:172; amino acid residues 6-181 of SEQ ID NO:173 which is encoded bynucleotides 16-543 of SEQ ID NO:172; amino acid residues 6-180 of SEQ IDNO:173 which is encoded by nucleotides 16-540 of SEQ ID NO:172; aminoacid residues 6-179 of SEQ ID NO:173 which is encoded by nucleotides16-537 of SEQ ID NO:172; amino acid residues 6-178 of SEQ ID NO:173which is encoded by nucleotides 16-534 of SEQ ID NO:172; amino acidresidues 6-177 of SEQ ID NO:173 which is encoded by nucleotides 16-531of SEQ ID NO:172; amino acid residues 6-176 of SEQ ID NO:173 which isencoded by nucleotides 16-528 of SEQ ID NO:172; amino acid residues6-175 of SEQ ID NO:173 which is encoded by nucleotides 16-525 of SEQ IDNO:172; amino acid residues 6-174 of SEQ ID NO:173 which is encoded bynucleotides 16-522 of SEQ ID NO:172; amino acid residues 6-173 of SEQ IDNO:173 which is encoded by nucleotides 16-519 of SEQ ID NO:172; aminoacid residues 6-172 of SEQ ID NO:173 which is encoded by nucleotides16-516 of SEQ ID NO:172; amino acid residues 7-182 of SEQ ID NO:173which is encoded by nucleotides 19-546 of SEQ ID NO:172; amino acidresidues 7-181 of SEQ ID NO:173 which is encoded by nucleotides 19-543of SEQ ID NO:172; amino acid residues 7-180 of SEQ ID NO:173 which isencoded by nucleotides 19-540 of SEQ ID NO:172; amino acid residues7-179 of SEQ ID NO:173 which is encoded by nucleotides 19-537 of SEQ IDNO:172; amino acid residues 7-178 of SEQ ID NO:173 which is encoded bynucleotides 19-534 of SEQ ID NO:172; amino acid residues 7-177 of SEQ IDNO:173 which is encoded by nucleotides 19-531 of SEQ ID NO:172; aminoacid residues 7-176 of SEQ ID NO:173 which is encoded by nucleotides19-528 of SEQ ID NO:172; amino acid residues 7-175 of SEQ ID NO:173which is encoded by nucleotides 19-525 of SEQ ID NO:172; amino acidresidues 7-174 of SEQ ID NO:173 which is encoded by nucleotides 19-522of SEQ ID NO:172; amino acid residues 7-173 of SEQ ID NO:173 which isencoded by nucleotides 19-519 of SEQ ID NO:172; amino acid residues7-172 of SEQ ID NO:173 which is encoded by nucleotides 19-516 of SEQ IDNO:172; amino acid residues 8-182 of SEQ ID NO:173 which is encoded bynucleotides 22-546 of SEQ ID NO:172; amino acid residues 8-181 of SEQ IDNO:173 which is encoded by nucleotides 22-543 of SEQ ID NO:172; aminoacid residues 8-180 of SEQ ID NO:173 which is encoded by nucleotides22-540 of SEQ ID NO:172; amino acid residues 8-179 of SEQ ID NO:173which is encoded by nucleotides 22-537 of SEQ ID NO:172; amino acidresidues 8-178 of SEQ ID NO:173 which is encoded by nucleotides 22-534of SEQ ID NO:172; amino acid residues 8-177 of SEQ ID NO:173 which isencoded by nucleotides 22-531 of SEQ ID NO:172; amino acid residues8-176 of SEQ ID NO:173 which is encoded by nucleotides 22-528 of SEQ IDNO:172; amino acid residues 8-175 of SEQ ID NO:173 which is encoded bynucleotides 22-525 of SEQ ID NO:172; amino acid residues 8-174 of SEQ IDNO:173 which is encoded by nucleotides 22-522 of SEQ ID NO:172; aminoacid residues 8-173 of SEQ ID NO:173 which is encoded by nucleotides22-519 of SEQ ID NO:172; amino acid residues 8-172 of SEQ ID NO:173which is encoded by nucleotides 22-516 of SEQ ID NO:172; amino acidresidues 9-182 of SEQ ID NO:173 which is encoded by nucleotides 25-546of SEQ ID NO:172; amino acid residues 9-181 of SEQ ID NO:173 which isencoded by nucleotides 25-543 of SEQ ID NO:172; amino acid residues9-180 of SEQ ID NO:173 which is encoded by nucleotides 25-540 of SEQ IDNO:172; amino acid residues 9-179 of SEQ ID NO:173 which is encoded bynucleotides 25-537 of SEQ ID NO:172; amino acid residues 9-178 of SEQ IDNO:173 which is encoded by nucleotides 25-534 of SEQ ID NO:172; aminoacid residues 9-177 of SEQ ID NO:173 which is encoded by nucleotides25-531 of SEQ ID NO:172; amino acid residues 9-176 of SEQ ID NO:173which is encoded by nucleotides 25-528 of SEQ ID NO:172; amino acidresidues 9-175 of SEQ ID NO:173 which is encoded by nucleotides 25-525of SEQ ID NO:172; amino acid residues 9-174 of SEQ ID NO:173 which isencoded by nucleotides 25-522 of SEQ ID NO:172; amino acid residues9-173 of SEQ ID NO:173 which is encoded by nucleotides 25-519 of SEQ IDNO:172; amino acid residues 9-172 of SEQ ID NO:173 which is encoded bynucleotides 25-516 of SEQ ID NO:172; amino acid residues 10-182 of SEQID NO:173 which is encoded by nucleotides 28-546 of SEQ ID NO:172; aminoacid residues 10-181 of SEQ ID NO:173 which is encoded by nucleotides28-543 of SEQ ID NO:172; amino acid residues 10-180 of SEQ ID NO:173which is encoded by nucleotides 28-540 of SEQ ID NO:172; amino acidresidues 10-179 of SEQ ID NO:173 which is encoded by nucleotides 28-537of SEQ ID NO:172; amino acid residues 10-178 of SEQ ID NO:173 which isencoded by nucleotides 28-534 of SEQ ID NO:172; amino acid residues10-177 of SEQ ID NO:173 which is encoded by nucleotides 28-531 of SEQ IDNO:172; amino acid residues 10-176 of SEQ ID NO:173 which is encoded bynucleotides 28-528 of SEQ ID NO:172; amino acid residues 10-175 of SEQID NO:173 which is encoded by nucleotides 28-525 of SEQ ID NO:172; aminoacid residues 10-174 of SEQ ID NO:173 which is encoded by nucleotides28-522 of SEQ ID NO:172; amino acid residues 10-173 of SEQ ID NO:173which is encoded by nucleotides 28-519 of SEQ ID NO:172; amino acidresidues 10-172 of SEQ ID NO:173 which is encoded by nucleotides 28-516of SEQ ID NO:172; amino acid residues 11-182 of SEQ ID NO:173 which isencoded by nucleotides 31-546 of SEQ ID NO:172; amino acid residues11-181 of SEQ ID NO:173 which is encoded by nucleotides 31-543 of SEQ IDNO:172; amino acid residues 11-180 of SEQ ID NO:173 which is encoded bynucleotides 31-540 of SEQ ID NO:172; amino acid residues 11-179 of SEQID NO:173 which is encoded by nucleotides 31-537 of SEQ ID NO:172; aminoacid residues 11-178 of SEQ ID NO:173 which is encoded by nucleotides31-534 of SEQ ID NO:172; amino acid residues 11-177 of SEQ ID NO:173which is encoded by nucleotides 31-531 of SEQ ID NO:172; amino acidresidues 11-176 of SEQ ID NO:173 which is encoded by nucleotides 31-528of SEQ ID NO:172; amino acid residues 11-175 of SEQ ID NO:173 which isencoded by nucleotides 31-525 of SEQ ID NO:172; amino acid residues11-174 of SEQ ID NO:173 which is encoded by nucleotides 31-522 of SEQ IDNO:172; amino acid residues 11-173 of SEQ ID NO:173 which is encoded bynucleotides 31-519 of SEQ ID NO:172; amino acid residues 11-172 of SEQID NO:173 which is encoded by nucleotides 31-516 of SEQ ID NO:172; aminoacid residues 12-182 of SEQ ID NO:173 which is encoded by nucleotides34-546 of SEQ ID NO:172; amino acid residues 12-181 of SEQ ID NO:173which is encoded by nucleotides 34-543 of SEQ ID NO:172; amino acidresidues 12-180 of SEQ ID NO:173 which is encoded by nucleotides 34-540of SEQ ID NO:172; amino acid residues 12-179 of SEQ ID NO:173 which isencoded by nucleotides 34-537 of SEQ ID NO:172; amino acid residues12-178 of SEQ ID NO:173 which is encoded by nucleotides 34-534 of SEQ IDNO:172; amino acid residues 12-177 of SEQ ID NO:173 which is encoded bynucleotides 34-531 of SEQ ID NO:172; amino acid residues 12-176 of SEQID NO:173 which is encoded by nucleotides 34-528 of SEQ ID NO:172; aminoacid residues 12-175 of SEQ ID NO:173 which is encoded by nucleotides34-525 of SEQ ID NO:172; amino acid residues 12-174 of SEQ ID NO:173which is encoded by nucleotides 34-522 of SEQ ID NO:172; amino acidresidues 12-173 of SEQ ID NO:173 which is encoded by nucleotides 34-519of SEQ ID NO:172; amino acid residues 12-172 of SEQ ID NO:173 which isencoded by nucleotides 34-516 of SEQ ID NO:172; amino acid residues13-182 of SEQ ID NO:173 which is encoded by nucleotides 37-546 of SEQ IDNO:172; amino acid residues 13-181 of SEQ ID NO:173 which is encoded bynucleotides 37-543 of SEQ ID NO:172; amino acid residues 13-180 of SEQID NO:173 which is encoded by nucleotides 37-540 of SEQ ID NO:172; aminoacid residues 13-179 of SEQ ID NO:173 which is encoded by nucleotides37-537 of SEQ ID NO:172; amino acid residues 13-178 of SEQ ID NO:173which is encoded by nucleotides 37-534 of SEQ ID NO:172; amino acidresidues 13-177 of SEQ ID NO:173 which is encoded by nucleotides 37-531of SEQ ID NO:172; amino acid residues 13-176 of SEQ ID NO:173 which isencoded by nucleotides 37-528 of SEQ ID NO:172; amino acid residues13-175 of SEQ ID NO:173 which is encoded by nucleotides 37-525 of SEQ IDNO:172; amino acid residues 13-174 of SEQ ID NO:173 which is encoded bynucleotides 37-522 of SEQ ID NO:172; amino acid residues 13-173 of SEQID NO:173 which is encoded by nucleotides 37-519 of SEQ ID NO:172; aminoacid residues 13-172 of SEQ ID NO:173 which is encoded by nucleotides37-516 of SEQ ID NO:172; amino acid residues 14-182 of SEQ ID NO:173which is encoded by nucleotides 40-546 of SEQ ID NO:172; amino acidresidues 14-181 of SEQ ID NO:173 which is encoded by nucleotides 40-543of SEQ ID NO:172; amino acid residues 14-180 of SEQ ID NO:173 which isencoded by nucleotides 40-540 of SEQ ID NO:172; amino acid residues14-179 of SEQ ID NO:173 which is encoded by nucleotides 40-537 of SEQ IDNO:172; amino acid residues 14-178 of SEQ ID NO:173 which is encoded bynucleotides 40-534 of SEQ ID NO:172; amino acid residues 14-177 of SEQID NO:173 which is encoded by nucleotides 40-531 of SEQ ID NO:172; aminoacid residues 14-176 of SEQ ID NO:173 which is encoded by nucleotides40-528 of SEQ ID NO:172; amino acid residues 14-175 of SEQ ID NO:173which is encoded by nucleotides 40-525 of SEQ ID NO:172; amino acidresidues 14-174 of SEQ ID NO:173 which is encoded by nucleotides 40-522of SEQ ID NO:172; amino acid residues 40-173 of SEQ ID NO:173 which isencoded by nucleotides 40-519 of SEQ ID NO:172; amino acid residues14-172 of SEQ ID NO:173 which is encoded by nucleotides 40-516 of SEQ IDNO:172; amino acid residues 15-182 of SEQ ID NO:173 which is encoded bynucleotides 43-546 of SEQ ID NO:172; amino acid residues 15-181 of SEQID NO:173 which is encoded by nucleotides 43-543 of SEQ ID NO:172; aminoacid residues 15-180 of SEQ ID NO:173 which is encoded by nucleotides43-540 of SEQ ID NO:172; amino acid residues 15-179 of SEQ ID NO:173which is encoded by nucleotides 43-537 of SEQ ID NO:172; amino acidresidues 15-178 of SEQ ID NO:173 which is encoded by nucleotides 43-534of SEQ ID NO:172; amino acid residues 15-177 of SEQ ID NO:173 which isencoded by nucleotides 43-531 of SEQ ID NO:172; amino acid residues15-176 of SEQ ID NO:173 which is encoded by nucleotides 43-528 of SEQ IDNO:172; amino acid residues 15-175 of SEQ ID NO:173 which is encoded bynucleotides 43-525 of SEQ ID NO:172; amino acid residues 15-174 of SEQID NO:173 which is encoded by nucleotides 43-522 of SEQ ID NO:172; aminoacid residues 15-173 of SEQ ID NO:173 which is encoded by nucleotides43-519 of SEQ ID NO:172; amino acid residues 15-172 of SEQ ID NO:173which is encoded by nucleotides 43-516 of SEQ ID NO:172; amino acidresidues 16-182 of SEQ ID NO:173 which is encoded by nucleotides 46-546of SEQ ID NO:172; amino acid residues 16-181 of SEQ ID NO:173 which isencoded by nucleotides 46-543 of SEQ ID NO:172; amino acid residues16-180 of SEQ ID NO:173 which is encoded by nucleotides 46-540 of SEQ IDNO:172; amino acid residues 16-179 of SEQ ID NO:173 which is encoded bynucleotides 46-537 of SEQ ID NO:172; amino acid residues 16-178 of SEQID NO:173 which is encoded by nucleotides 46-534 of SEQ ID NO:172; aminoacid residues 16-177 of SEQ ID NO:173 which is encoded by nucleotides46-531 of SEQ ID NO:172; amino acid residues 16-176 of SEQ ID NO:173which is encoded by nucleotides 46-528 of SEQ ID NO:172; amino acidresidues16-175 of SEQ ID NO:173 which is encoded by nucleotides 46-525of SEQ ID NO:172; amino acid residues 16-174 of SEQ ID NO:173 which isencoded by nucleotides 46-522 of SEQ ID NO:172; amino acid residues16-173 of SEQ ID NO:173 which is encoded by nucleotides 46-519 of SEQ IDNO:172; and amino acid residues 16-172 of SEQ ID NO:173 which is encodedby nucleotides 46-516 of SEQ ID NO:172. The N-terminally andC-terminally modified biologically active mutants of IL-29 C5 mutants ofthe present invention may also include an N-terminal Methione ifexpressed, for instance, in E. coli.

Additional IL-29 C5 N-terminally and C-terminally biologically activemutants include, for example, amino acid residues 2-176 of SEQ ID NO:175which is encoded by nucleotides 4-528 of SEQ ID NO:174; amino acidresidues 2-175 of SEQ ID NO:175 which is encoded by nucleotides 4-525 ofSEQ ID NO:174; amino acid residues 2-174 of SEQ ID NO:175 which isencoded by nucleotides 4-522 of SEQ ID NO:174; amino acid residues 2-173of SEQ ID NO:175 which is encoded by nucleotides 4-519 of SEQ ID NO:174;amino acid residues 2-172 of SEQ ID NO:175 which is encoded bynucleotides 4-516 of SEQ ID NO:174; amino acid residues 2-171 of SEQ IDNO:175 which is encoded by nucleotides 4-513 of SEQ ID NO:174; aminoacid residues 2-170 of SEQ ID NO:175 which is encoded by nucleotides4-510 of SEQ ID NO:174; amino acid residues 2-169 of SEQ ID NO:175 whichis encoded by nucleotides 4-507 of SEQ ID NO:174; amino acid residues2-168 of SEQ ID NO:175 which is encoded by nucleotides 4-504 of SEQ IDNO:174; amino acid residues 2-167 of SEQ ID NO:175 which is encoded bynucleotides 4-501 of SEQ ID NO:174; amino acid residues 2-166 of SEQ IDNO:175 which is encoded by nucleotides 4-498 of SEQ ID NO:174; aminoacid residues 3-176 of SEQ ID NO:175 which is encoded by nucleotides7-528 of SEQ ID NO:174; amino acid residues 3-175 of SEQ ID NO:175 whichis encoded by nucleotides 7-525 of SEQ ID NO:174; amino acid residues3-174 of SEQ ID NO:175 which is encoded by nucleotides 7-522 of SEQ IDNO:174; amino acid residues 3-173 of SEQ ID NO:175 which is encoded bynucleotides 7-519 of SEQ ID NO:174; amino acid residues 3-172 of SEQ IDNO:175 which is encoded by nucleotides 7-516 of SEQ ID NO:174; aminoacid residues 3-171 of SEQ ID NO:175 which is encoded by nucleotides7-513 of SEQ ID NO:174; amino acid residues 3-170 of SEQ ID NO:175 whichis encoded by nucleotides 7-510 of SEQ ID NO:174; amino acid residues3-169 of SEQ ID NO:175 which is encoded by nucleotides 7-507 of SEQ IDNO:174; amino acid residues 3-168 of SEQ ID NO:175 which is encoded bynucleotides 7-504 of SEQ ID NO:174; amino acid residues 3-167 of SEQ IDNO:175 which is encoded by nucleotides 7-501 of SEQ ID NO:174; aminoacid residues 3-166 of SEQ ID NO:175 which is encoded by nucleotides7-498 of SEQ ID NO:174; amino acid residues 4-176 of SEQ ID NO:175 whichis encoded by nucleotides 10-528 of SEQ ID NO:174; amino acid residues4-175 of SEQ ID NO:175 which is encoded by nucleotides 10-525 of SEQ IDNO:174; amino acid residues 4-174 of SEQ ID NO:175 which is encoded bynucleotides 10-522 of SEQ ID NO:174; amino acid residues 4-173 of SEQ IDNO:175 which is encoded by nucleotides 10-519 of SEQ ID NO:174; aminoacid residues 4-172 of SEQ ID NO:175 which is encoded by nucleotides10-516 of SEQ ID NO:174; amino acid residues 4-171 of SEQ ID NO:175which is encoded by nucleotides 10-513 of SEQ ID NO:174; amino acidresidues 4-170 of SEQ ID NO:175 which is encoded by nucleotides 10-510of SEQ ID NO:174; amino acid residues 4-169 of SEQ ID NO:175 which isencoded by nucleotides 10-507 of SEQ ID NO:174; amino acid residues4-168 of SEQ ID NO:175 which is encoded by nucleotides 10-504 of SEQ IDNO:174; amino acid residues 4-167 of SEQ ID NO:175 which is encoded bynucleotides 10-501 of SEQ ID NO:174; amino acid residues 4-166 of SEQ IDNO:175 which is encoded by nucleotides 10-498 of SEQ ID NO:174; aminoacid residues 5-176 of SEQ ID NO:175 which is encoded by nucleotides13-528 of SEQ ID NO:174; amino acid residues 5-175 of SEQ ID NO:175which is encoded by nucleotides 13-525 of SEQ ID NO:174; amino acidresidues 5-174 of SEQ ID NO:175 which is encoded by nucleotides 13-522of SEQ ID NO:174; amino acid residues 5-173 of SEQ ID NO:175 which isencoded by nucleotides 13-519 of SEQ ID NO:174; amino acid residues5-172 of SEQ ID NO:175 which is encoded by nucleotides 13-516 of SEQ IDNO:174; amino acid residues 5-171 of SEQ ID NO:175 which is encoded bynucleotides 13-513 of SEQ ID NO:174; amino acid residues 5-170 of SEQ IDNO:175 which is encoded by nucleotides 13-510 of SEQ ID NO:174; aminoacid residues 5-169 of SEQ ID NO:175 which is encoded by nucleotides13-507 of SEQ ID NO:174; amino acid residues 5-168 of SEQ ID NO:175which is encoded by nucleotides 13-504 of SEQ ID NO:174; amino acidresidues 5-167 of SEQ ID NO:175 which is encoded by nucleotides 13-501of SEQ ID NO:174; amino acid residues 5-166 of SEQ ID NO:175 which isencoded by nucleotides 13-498 of SEQ ID NO:174; amino acid residues6-176 of SEQ ID NO:175 which is encoded by nucleotides 16-528 of SEQ IDNO:174; amino acid residues 6-175 of SEQ ID NO:175 which is encoded bynucleotides 16-525 of SEQ ID NO:174; amino acid residues 6-174 of SEQ IDNO:175 which is encoded by nucleotides 16-522 of SEQ ID NO:174; aminoacid residues 6-173 of SEQ ID NO:175 which is encoded by nucleotides16-519 of SEQ ID NO:174; amino acid residues 6-172 of SEQ ID NO:175which is encoded by nucleotides 16-516 of SEQ ID NO:174; amino acidresidues 6-171 of SEQ ID NO:175 which is encoded by nucleotides 16-513of SEQ ID NO:174; amino acid residues 6-170 of SEQ ID NO:175 which isencoded by nucleotides 16-510 of SEQ ID NO:174; amino acid residues6-169 of SEQ ID NO:175 which is encoded by nucleotides 16-507 of SEQ IDNO:174; amino acid residues 6-168 of SEQ ID NO:175 which is encoded bynucleotides 16-504 of SEQ ID NO:174; amino acid residues 6-167 of SEQ IDNO:175 which is encoded by nucleotides 16-501 of SEQ ID NO:174; aminoacid residues 6-166 of SEQ ID NO:175 which is encoded by nucleotides16-498 of SEQ ID NO:174; amino acid residues 7-176 of SEQ ID NO:175which is encoded by nucleotides 19-528 of SEQ ID NO:174; amino acidresidues 7-175 of SEQ ID NO:175 which is encoded by nucleotides 19-525of SEQ ID NO:174; amino acid residues 7-174 of SEQ ID NO:175 which isencoded by nucleotides 19-522 of SEQ ID NO:174; amino acid residues7-173 of SEQ ID NO:175 which is encoded by nucleotides 19-519 of SEQ IDNO:174; amino acid residues 7-172 of SEQ ID NO:175 which is encoded bynucleotides 19-516 of SEQ ID NO:174; amino acid residues 7-171 of SEQ IDNO:175 which is encoded by nucleotides 19-513 of SEQ ID NO:174; aminoacid residues 7-170 of SEQ ID NO:175 which is encoded by nucleotides19-510 of SEQ ID NO:174; amino acid residues 7-169 of SEQ ID NO:175which is encoded by nucleotides 19-507 of SEQ ID NO:174; amino acidresidues 7-168 of SEQ ID NO:175 which is encoded by nucleotides 19-504of SEQ ID NO:174; amino acid residues 7-167 of SEQ ID NO:175 which isencoded by nucleotides 19-501 of SEQ ID NO:174; amino acid residues7-166 of SEQ ID NO:175 which is encoded by nucleotides 19-498 of SEQ IDNO:174; amino acid residues 8-176 of SEQ ID NO:175 which is encoded bynucleotides 22-528 of SEQ ID NO:174; amino acid residues 8-175 of SEQ IDNO:175 which is encoded by nucleotides 22-525 of SEQ ID NO:174; aminoacid residues 8-174 of SEQ ID NO:175 which is encoded by nucleotides22-522 of SEQ ID NO:174; amino acid residues 8-173 of SEQ ID NO:175which is encoded by nucleotides 22-519 of SEQ ID NO:174; amino acidresidues 8-172 of SEQ ID NO:175 which is encoded by nucleotides 22-516of SEQ ID NO:174; amino acid residues 8-171 of SEQ ID NO:175 which isencoded by nucleotides 22-513 of SEQ ID NO:174; amino acid residues8-170 of SEQ ID NO:175 which is encoded by nucleotides 22-510 of SEQ IDNO:174; amino acid residues 8-169 of SEQ ID NO:175 which is encoded bynucleotides 22-507 of SEQ ID NO:174; amino acid residues 8-168 of SEQ IDNO:175 which is encoded by nucleotides 22-504 of SEQ ID NO:174; aminoacid residues 8-167 of SEQ ID NO:175 which is encoded by nucleotides22-501 of SEQ ID NO:174; amino acid residues 8-166 of SEQ ID NO:175which is encoded by nucleotides 22-498 of SEQ ID NO:174; amino acidresidues 9-176 of SEQ ID NO:175 which is encoded by nucleotides 25-528of SEQ ID NO:174; amino acid residues 9-175 of SEQ ID NO:175 which isencoded by nucleotides 25-525 of SEQ ID NO:174; amino acid residues9-174 of SEQ ID NO:175 which is encoded by nucleotides 25-522 of SEQ IDNO:174; amino acid residues 9-173 of SEQ ID NO:175 which is encoded bynucleotides 25-519 of SEQ ID NO:174; amino acid residues 9-172 of SEQ IDNO:175 which is encoded by nucleotides 25-516 of SEQ ID NO:174; aminoacid residues 9-171 of SEQ ID NO:175 which is encoded by nucleotides25-513 of SEQ ID NO:174; amino acid residues 9-170 of SEQ ID NO:175which is encoded by nucleotides 25-510 of SEQ ID NO:174; amino acidresidues 9-169 of SEQ ID NO:175 which is encoded by nucleotides 25-507of SEQ ID NO:174; amino acid residues 9-168 of SEQ ID NO:175 which isencoded by nucleotides 25-504 of SEQ ID NO:174; amino acid residues9-167 of SEQ ID NO:175 which is encoded by nucleotides 25-501 of SEQ IDNO:174; amino acid residues 9-166 of SEQ ID NO:175 which is encoded bynucleotides 25-498 of SEQ ID NO:174; amino acid residues 10-176 of SEQID NO:175 which is encoded by nucleotides 28-528 of SEQ ID NO:174; aminoacid residues 10-175 of SEQ ID NO:175 which is encoded by nucleotides28-525 of SEQ ID NO:174; amino acid residues 10-174 of SEQ ID NO:175which is encoded by nucleotides 28-522 of SEQ ID NO:174; amino acidresidues 10-173 of SEQ ID NO:175 which is encoded by nucleotides 28-519of SEQ ID NO:174; amino acid residues 10-172 of SEQ ID NO:175 which isencoded by nucleotides 28-516 of SEQ ID NO:174; amino acid residues10-171 of SEQ ID NO:175 which is encoded by nucleotides 28-513 of SEQ IDNO:174; amino acid residues 10-170 of SEQ ID NO:175 which is encoded bynucleotides 28-510 of SEQ ID NO:174; amino acid residues 10-169 of SEQID NO:175 which is encoded by nucleotides 28-507 of SEQ ID NO:174; aminoacid residues 10-168 of SEQ ID NO:175 which is encoded by nucleotides28-504 of SEQ ID NO:174; amino acid residues 10-167 of SEQ ID NO:175which is encoded by nucleotides 28-501 of SEQ ID NO:174; and amino acidresidues 10-166 of SEQ ID NO:175 which is encoded by nucleotides 28-498of SEQ ID NO:174. The N-terminally and C-terminally modifiedbiologically active mutants of IL-29 C5 mutants of the present inventionmay also include an N-terminal Methione if expressed, for instance, inE. coli.

In addition to the IL-29 C5 mutants, the present invention also includesIL-29 polypeptides comprising a mutation at the first cysteine position,C1, of the mature polypeptide. For example, C1 from the N-terminus ofthe polypeptide of SEQ ID NO:4, is the cysteine at position 15, orposition 16 (additional N-terminal Met) if expressed in E. coli (see,for example, SEQ ID NO:15). These IL-29 C1 mutant polypeptides will thushave a predicted disulfide bond pattern of C2(Cys49 of SEQ IDNO:4)/C4(Cys145 of SEQ ID NO:4) and C3(Cys112 of SEQ ID NO:4)/C5(Cys171of SEQ ID NO:4). Additional IL-29 C1 mutant molecules of the presentinvention include polynucleotide molecules as shown in SEQ ID NOs:74,76, 78, and 80, including DNA and RNA molecules, that encode IL-29 C1mutant polypeptides as shown in SEQ ID NOs:75, 77, 79 and 81,respectively. Additional IL-29 C1 mutant molecules of the presentinvention include polynucleotide molecules as shown in SEQ ID NOs:90,92, 98, and 100, including DNA and RNA molecules, that encode IL-29 C1mutant polypeptides as shown in SEQ ID NOs:91, 93, 99, and 101,respectively (PCT publication WO 03/066002 (Kotenko et al.)).Additional, IL-29 C1 mutant molecules of the present invention includepolynucleotide molecules as shown in SEQ ID NOs:106, 108, 114, and 116,including DNA and RNA molecules, that encode IL-29 C1 mutantpolypeptides as shown in SEQ ID NOs:107, 109, 115, and 117, respectively(PCT publication WO 02/092762 (Baum et al.)).

The present invention also includes biologically active mutants of IL-29C1 cysteine mutants which provide, at least partially, anti-tumoractivity and/or immune regulatory activity. The first cysteine or C1from the N-terminus of IL-29 can mutated to any amino acid that does notform a disulfide bond with another cysteine, e.g., serine, alanine,threonine, valine or aspargine. The biologically active mutants of IL-29C1 cysteine mutants of the present invention include N-, C-, and N- andC-terminal deletions of IL-29, e.g., the polypeptide of SEQ ID NOs:171encoded by the polynucleotide of SEQ ID NO:170.

N-terminally modified biologically active mutants of IL-29 C1 mutantsinclude, for example, amino acid residues 2-182 of SEQ ID NO:171 whichis encoded by nucleotides 4-546 of SEQ ID NO:170; amino acid residues3-182 of SEQ ID NO:171 which is encoded by nucleotides 7-546 of SEQ IDNO:170; amino acid residues 4-182 of SEQ ID NO:171 which is encoded bynucleotides 10-546 of SEQ ID NO:170; amino acid residues 5-182 of SEQ IDNO:171 which is encoded by nucleotides 13-546 of SEQ ID NO:170; aminoacid residues 6-182 of SEQ ID NO:171 which is encoded by nucleotides16-546 of SEQ ID NO:170; amino acid residues 7-182 of SEQ ID NO:171which is encoded by nucleotides 19-546 of SEQ ID NO:170; amino acidresidues 8-182 of SEQ ID NO:171 which is encoded by nucleotides 22-546of SEQ ID NO:170; amino acid residues 9-182 of SEQ ID NO:171 which isencoded by nucleotides 25-546 of SEQ ID NO:170; amino acid residues10-182 of SEQ ID NO:171 which is encoded by nucleotides 28-546 of SEQ IDNO:170; amino acid residues 11-182 of SEQ ID NO:171 which is encoded bynucleotides 31-546 of SEQ ID NO:170; amino acid residues 12-182 of SEQID NO:171 which is encoded by nucleotides 34-182 of SEQ ID NO:170; aminoacid residues 13-182 of SEQ ID NO:171 which is encoded by nucleotides37-546 of SEQ ID NO:170; amino acid residues 14-182 of SEQ ID NO:171which is encoded by nucleotides 40-546 of SEQ ID NO:170; amino acidresidues 15-182 of SEQ ID NO:171 which is encoded by nucleotides 43-546of SEQ ID NO:170; and amino acid residues 16-182 of SEQ ID NO:171 whichis encoded by nucleotides 46-546 of SEQ ID NO:170. The N-terminallymodified biologically active mutants of IL-29 C1 mutants of the presentinvention may also include an N-terminal Methione if expressed, forinstance, in E. coli.

C-terminally modified biologically active mutants of IL-29 C1 mutantsinclude, for example, amino acid residues 1-181 of SEQ ID NO:171 whichis encoded by nucleotides 1-543 of SEQ ID NO:170; amino acid residues1-180 of SEQ ID NO:171 which is encoded by nucleotides 1-540 of SEQ IDNO:170; amino acid residues 1-179 of SEQ ID NO:171 which is encoded bynucleotides 1-537 of SEQ ID NO:170; amino acid residues 1-178 of SEQ IDNO:171 which is encoded by nucleotides 1-534 of SEQ ID NO:170; aminoacid residues 1-177 of SEQ ID NO:171 which is encoded by nucleotides1-531 of SEQ ID NO:170; amino acid residues 1-176 of SEQ ID NO:171 whichis encoded by nucleotides 1-528 of SEQ ID NO:170; amino acid residues1-175 of SEQ ID NO:171 which is encoded by nucleotides 1-525 of SEQ IDNO:170; amino acid residues 1-174 of SEQ ID NO:171 which is encoded bynucleotides 1-522 of SEQ ID NO:170; amino acid residues 1-173 of SEQ IDNO:171 which is encoded by nucleotides 1-519 of SEQ ID NO:170; and aminoacid residues 1-172 of SEQ ID NO:171 which is encoded by nucleotides1-516 of SEQ ID NO:170.

N-terminally and C-terminally modified biologically active mutants ofIL-29 C1 mutants include, for example, amino acid residues 2-181 of SEQID NO:171 which is encoded by nucleotides 4-543 of SEQ ID NO:170; aminoacid residues 2-180 of SEQ ID NO:171 which is encoded by nucleotides4-540 of SEQ ID NO:170; amino acid residues 2-179 of SEQ ID NO:171 whichis encoded by nucleotides 4-537 of SEQ ID NO:170; amino acid residues2-178 of SEQ ID NO:171 which is encoded by nucleotides 4-534 of SEQ IDNO:170; amino acid residues 2-177 of SEQ ID NO:171 which is encoded bynucleotides 4-531 of SEQ ID NO:170; amino acid residues 2-176 of SEQ IDNO:171 which is encoded by nucleotides 4-528 of SEQ ID NO:170; aminoacid residues 2-175 of SEQ ID NO:171 which is encoded by nucleotides4-525 of SEQ ID NO:170; amino acid residues 2-174 of SEQ ID NO:171 whichis encoded by nucleotides 4-522 of SEQ ID NO:170; amino acid residues2-173 of SEQ ID NO:171 which is encoded by nucleotides 4-519 of SEQ IDNO:170; amino acid residues 2-172 of SEQ ID NO:171 which is encoded bynucleotides 4-516 of SEQ ID NO:170; amino acid residues 3-181 of SEQ IDNO:171 which is encoded by nucleotides 7-543 of SEQ ID NO:170; aminoacid residues 3-180 of SEQ ID NO:171 which is encoded by nucleotides7-540 of SEQ ID NO:170; amino acid residues 3-179 of SEQ ID NO:171 whichis encoded by nucleotides 7-537 of SEQ ID NO:170; amino acid residues3-178 of SEQ ID NO:171 which is encoded by nucleotides 7-534 of SEQ IDNO:170; amino acid residues 3-177 of SEQ ID NO:171 which is encoded bynucleotides 7-531 of SEQ ID NO:170; amino acid residues 3-176 of SEQ IDNO:171 which is encoded by nucleotides 7-528 of SEQ ID NO:170; aminoacid residues 3-175 of SEQ ID NO:171 which is encoded by nucleotides7-525 of SEQ ID NO:170; amino acid residues 3-174 of SEQ ID NO:171 whichis encoded by nucleotides 7-522 of SEQ ID NO:170; amino acid residues3-173 of SEQ ID NO:171 which is encoded by nucleotides 7-519 of SEQ IDNO:170; amino acid residues 3-172 of SEQ ID NO:171 which is encoded bynucleotides 7-516 of SEQ ID NO:170; amino acid residues 4-181 of SEQ IDNO:171 which is encoded by nucleotides 10-543 of SEQ ID NO:170; aminoacid residues 4-180 of SEQ ID NO:171 which is encoded by nucleotides10-540 of SEQ ID NO:170; amino acid residues 4-179 of SEQ ID NO:171which is encoded by nucleotides 10-537 of SEQ ID NO:170; amino acidresidues 4-178 of SEQ ID NO:171 which is encoded by nucleotides 10-534of SEQ ID NO:170; amino acid residues 4-177 of SEQ ID NO:171 which isencoded by nucleotides 10-531 of SEQ ID NO:170; amino acid residues4-176 of SEQ ID NO:171 which is encoded by nucleotides 10-528 of SEQ IDNO:170; amino acid residues 4-175 of SEQ ID NO:171 which is encoded bynucleotides 10-525 of SEQ ID NO:170; amino acid residues 4-174 of SEQ IDNO:171 which is encoded by nucleotides 10-522 of SEQ ID NO:170; aminoacid residues 4-173 of SEQ ID NO:171 which is encoded by nucleotides10-519 of SEQ ID NO:170; amino acid residues 4-172 of SEQ ID NO:171which is encoded by nucleotides 10-516 of SEQ ID NO:170; amino acidresidues 5-181 of SEQ ID NO:171 which is encoded by nucleotides 13-543of SEQ ID NO:170; amino acid residues 5-180 of SEQ ID NO:171 which isencoded by nucleotides 13-540 of SEQ ID NO:170; amino acid residues5-179 of SEQ ID NO:171 which is encoded by nucleotides 13-537 of SEQ IDNO:170; amino acid residues 5-178 of SEQ ID NO:171 which is encoded bynucleotides 13-534 of SEQ ID NO:170; amino acid residues 5-177 of SEQ IDNO:171 which is encoded by nucleotides 13-531 of SEQ ID NO:170; aminoacid residues 5-176 of SEQ ID NO:171 which is encoded by nucleotides13-528 of SEQ ID NO:170; amino acid residues 5-175 of SEQ ID NO:171which is encoded by nucleotides 13-525 of SEQ ID NO:170; amino acidresidues 5-174 of SEQ ID NO:171 which is encoded by nucleotides 13-522of SEQ ID NO:170; amino acid residues 5-173 of SEQ ID NO:171 which isencoded by nucleotides 13-519 of SEQ ID NO:170; amino acid residues5-172 of SEQ ID NO:171 which is encoded by nucleotides 13-516 of SEQ IDNO:170; amino acid residues 6-181 of SEQ ID NO:171 which is encoded bynucleotides 16-543 of SEQ ID NO:170; amino acid residues 6-180 of SEQ IDNO:171 which is encoded by nucleotides 16-540 of SEQ ID NO:170; aminoacid residues 6-179 of SEQ ID NO:171 which is encoded by nucleotides16-537 of SEQ ID NO:170; amino acid residues 6-178 of SEQ ID NO:171which is encoded by nucleotides 16-534 of SEQ ID NO:170; amino acidresidues 6-177 of SEQ ID NO:171 which is encoded by nucleotides 16-531of SEQ ID NO:170; amino acid residues 6-176 of SEQ ID NO:171 which isencoded by nucleotides 16-528 of SEQ ID NO:170; amino acid residues6-175 of SEQ ID NO:171 which is encoded by nucleotides 16-525 of SEQ IDNO:170; amino acid residues 6-174 of SEQ ID NO:171 which is encoded bynucleotides 16-522 of SEQ ID NO:170; amino acid residues 6-173 of SEQ IDNO:171 which is encoded by nucleotides 16-519 of SEQ ID NO:170; aminoacid residues 6-172 of SEQ ID NO:171 which is encoded by nucleotides16-516 of SEQ ID NO:170; amino acid residues 7-181 of SEQ ID NO:171which is encoded by nucleotides 19-543 of SEQ ID NO:170; amino acidresidues 7-180 of SEQ ID NO:171 which is encoded by nucleotides 19-540of SEQ ID NO:170; amino acid residues 7-179 of SEQ ID NO:171 which isencoded by nucleotides 19-537 of SEQ ID NO:170; amino acid residues7-178 of SEQ ID NO:171 which is encoded by nucleotides 19-534 of SEQ IDNO:170; amino acid residues 7-177 of SEQ ID NO:171 which is encoded bynucleotides 19-531 of SEQ ID NO:170; amino acid residues 7-176 of SEQ IDNO:171 which is encoded by nucleotides 19-528 of SEQ ID NO:170; aminoacid residues 7-175 of SEQ ID NO:171 which is encoded by nucleotides19-525 of SEQ ID NO:170; amino acid residues 7-174 of SEQ ID NO:171which is encoded by nucleotides 19-522 of SEQ ID NO:170; amino acidresidues 7-173 of SEQ ID NO:171 which is encoded by nucleotides 19-519of SEQ ID NO:170; amino acid residues 7-172 of SEQ ID NO:171 which isencoded by nucleotides 19-516 of SEQ ID NO:170; amino acid residues8-181 of SEQ ID NO:171 which is encoded by nucleotides 22-543 of SEQ IDNO:170; amino acid residues 8-180 of SEQ ID NO:171 which is encoded bynucleotides 22-540 of SEQ ID NO:170; amino acid residues 8-179 of SEQ IDNO:171 which is encoded by nucleotides 22-537 of SEQ ID NO:170; aminoacid residues 8-178 of SEQ ID NO:171 which is encoded by nucleotides22-534 of SEQ ID NO:170; amino acid residues 8-177 of SEQ ID NO:171which is encoded by nucleotides 22-531 of SEQ ID NO:170; amino acidresidues 8-176 of SEQ ID NO:171 which is encoded by nucleotides 22-528of SEQ ID NO:170; amino acid residues 8-175 of SEQ ID NO:171 which isencoded by nucleotides 22-525 of SEQ ID NO:170; amino acid residues8-174 of SEQ ID NO:171 which is encoded by nucleotides 22-522 of SEQ IDNO:170; amino acid residues 8-173 of SEQ ID NO:171 which is encoded bynucleotides 22-519 of SEQ ID NO:170; amino acid residues 8-172 of SEQ IDNO:171 which is encoded by nucleotides 22-516 of SEQ ID NO:170; aminoacid residues 9-181 of SEQ ID NO:171 which is encoded by nucleotides25-543 of SEQ ID NO:170; amino acid residues 9-180 of SEQ ID NO:171which is encoded by nucleotides 25-540 of SEQ ID NO:170; amino acidresidues 9-179 of SEQ ID NO:171 which is encoded by nucleotides 25-537of SEQ ID NO:170; amino acid residues 9-178 of SEQ ID NO:171 which isencoded by nucleotides 25-534 of SEQ ID NO:170; amino acid residues9-177 of SEQ ID NO:171 which is encoded by nucleotides 25-531 of SEQ IDNO:170; amino acid residues 9-176 of SEQ ID NO:171 which is encoded bynucleotides 25-528 of SEQ ID NO:170; amino acid residues 9-175 of SEQ IDNO:171 which is encoded by nucleotides 25-525 of SEQ ID NO:170; aminoacid residues 9-174 of SEQ ID NO:171 which is encoded by nucleotides25-522 of SEQ ID NO:170; amino acid residues 9-173 of SEQ ID NO:171which is encoded by nucleotides 25-519 of SEQ ID NO:170; amino acidresidues 9-172 of SEQ ID NO:171 which is encoded by nucleotides 25-516of SEQ ID NO:170; amino acid residues 10-181 of SEQ ID NO:171 which isencoded by nucleotides 28-543 of SEQ ID NO:170; amino acid residues10-180 of SEQ ID NO:171 which is encoded by nucleotides 28-540 of SEQ IDNO:170; amino acid residues 10-179 of SEQ ID NO:171 which is encoded bynucleotides 28-537 of SEQ ID NO:170; amino acid residues 10-178 of SEQID NO:171 which is encoded by nucleotides 28-534 of SEQ ID NO:170; aminoacid residues 10-177 of SEQ ID NO:171 which is encoded by nucleotides28-531 of SEQ ID NO:170; amino acid residues 10-176 of SEQ ID NO:171which is encoded by nucleotides 28-528 of SEQ ID NO:170; amino acidresidues 10-175 of SEQ ID NO:171 which is encoded by nucleotides 28-525of SEQ ID NO:170; amino acid residues 10-174 of SEQ ID NO:171 which isencoded by nucleotides 28-522 of SEQ ID NO:170; amino acid residues10-173 of SEQ ID NO:171 which is encoded by nucleotides 28-519 of SEQ IDNO:170; amino acid residues 10-172 of SEQ ID NO:171 which is encoded bynucleotides 28-516 of SEQ ID NO:170; amino acid residues 11-181 of SEQID NO:171 which is encoded by nucleotides 31-543 of SEQ ID NO:170; aminoacid residues 11-180 of SEQ ID NO:171 which is encoded by nucleotides31-540 of SEQ ID NO:170; amino acid residues 11-179 of SEQ ID NO:171which is encoded by nucleotides 31-537 of SEQ ID NO:170; amino acidresidues 11-178 of SEQ ID NO:171 which is encoded by nucleotides 31-534of SEQ ID NO:170; amino acid residues 11-177 of SEQ ID NO:171 which isencoded by nucleotides 31-531 of SEQ ID NO:170; amino acid residues11-176 of SEQ ID NO:171 which is encoded by nucleotides 31-528 of SEQ IDNO:170; amino acid residues 11-175 of SEQ ID NO:171 which is encoded bynucleotides 31-525 of SEQ ID NO:170; amino acid residues 11-174 of SEQID NO:171 which is encoded by nucleotides 31-522 of SEQ ID NO:170; aminoacid residues 11-173 of SEQ ID NO:171 which is encoded by nucleotides31-519 of SEQ ID NO:170; amino acid residues 11-172 of SEQ ID NO:171which is encoded by nucleotides 31-516 of SEQ ID NO:170; amino acidresidues 12-181 of SEQ ID NO:171 which is encoded by nucleotides 34-543of SEQ ID NO:170; amino acid residues 12-180 of SEQ ID NO:171 which isencoded by nucleotides 34-540 of SEQ ID NO:170; amino acid residues12-179 of SEQ ID NO:171 which is encoded by nucleotides 34-537 of SEQ IDNO:170; amino acid residues 12-178 of SEQ ID NO:171 which is encoded bynucleotides 34-534 of SEQ ID NO:170; amino acid residues 12-177 of SEQID NO:171 which is encoded by nucleotides 34-531 of SEQ ID NO:170; aminoacid residues 12-176 of SEQ ID NO:171 which is encoded by nucleotides34-528 of SEQ ID NO:170; amino acid residues 12-175 of SEQ ID NO:171which is encoded by nucleotides 34-525 of SEQ ID NO:170; amino acidresidues 12-174 of SEQ ID NO:171 which is encoded by nucleotides 34-522of SEQ ID NO:170; amino acid residues 12-173 of SEQ ID NO:171 which isencoded by nucleotides 34-519 of SEQ ID NO:170; amino acid residues12-172 of SEQ ID NO:171 which is encoded by nucleotides 34-516 of SEQ IDNO:170; amino acid residues 13-181 of SEQ ID NO:171 which is encoded bynucleotides 37-543 of SEQ ID NO:170; amino acid residues 13-180 of SEQID NO:171 which is encoded by nucleotides 37-540 of SEQ ID NO:170; aminoacid residues 13-179 of SEQ ID NO:171 which is encoded by nucleotides37-537 of SEQ ID NO:170; amino acid residues 13-178 of SEQ ID NO:171which is encoded by nucleotides 37-534 of SEQ ID NO:170; amino acidresidues 13-177 of SEQ ID NO:171 which is encoded by nucleotides 37-531of SEQ ID NO:170; amino acid residues 13-176 of SEQ ID NO:171 which isencoded by nucleotides 37-528 of SEQ ID NO:170; amino acid residues13-175 of SEQ ID NO:171 which is encoded by nucleotides 37-525 of SEQ IDNO:170; amino acid residues 13-174 of SEQ ID NO:171 which is encoded bynucleotides 37-522 of SEQ ID NO:170; amino acid residues 13-173 of SEQID NO:171 which is encoded by nucleotides 37-519 of SEQ ID NO:170; aminoacid residues 13-172 of SEQ ID NO:171 which is encoded by nucleotides37-516 of SEQ ID NO:170; amino acid residues 14-181 of SEQ ID NO:171which is encoded by nucleotides 40-543 of SEQ ID NO:170; amino acidresidues 14-180 of SEQ ID NO:171 which is encoded by nucleotides 40-540of SEQ ID NO:170; amino acid residues 14-179 of SEQ ID NO:171 which isencoded by nucleotides 40-537 of SEQ ID NO:170; amino acid residues14-178 of SEQ ID NO:171 which is encoded by nucleotides 40-534 of SEQ IDNO:170; amino acid residues 14-177 of SEQ ID NO:171 which is encoded bynucleotides 40-531 of SEQ ID NO:170; amino acid residues 14-176 of SEQID NO:171 which is encoded by nucleotides 40-528 of SEQ ID NO:170; aminoacid residues 14-175 of SEQ ID NO:171 which is encoded by nucleotides40-525 of SEQ ID NO:170; amino acid residues 14-174 of SEQ ID NO:171which is encoded by nucleotides 40-522 of SEQ ID NO:170; amino acidresidues 14-173 of SEQ ID NO:171 which is encoded by nucleotides 40-519of SEQ ID NO:170; amino acid residues 14-172 of SEQ ID NO:171 which isencoded by nucleotides 40-516 of SEQ ID NO:170; amino acid residues15-181 of SEQ ID NO:171 which is encoded by nucleotides 43-543 of SEQ IDNO:170; amino acid residues 15-180 of SEQ ID NO:171 which is encoded bynucleotides 43-540 of SEQ ID NO:170; amino acid residues 15-179 of SEQID NO:171 which is encoded by nucleotides 43-537 of SEQ ID NO:170; aminoacid residues 15-178 of SEQ ID NO:171 which is encoded by nucleotides43-534 of SEQ ID NO:170; amino acid residues 15-177 of SEQ ID NO:171which is encoded by nucleotides 43-531 of SEQ ID NO:170; amino acidresidues 15-176 of SEQ ID NO:171 which is encoded by nucleotides 43-528of SEQ ID NO:170; amino acid residues 15-175 of SEQ ID NO:171 which isencoded by nucleotides 43-525 of SEQ ID NO:170; amino acid residues15-174 of SEQ ID NO:171 which is encoded by nucleotides 43-522 of SEQ IDNO:170; amino acid residues 15-173 of SEQ ID NO:171 which is encoded bynucleotides 43-519 of SEQ ID NO:170; amino acid residues 15-172 of SEQID NO:171 which is encoded by nucleotides 43-516 of SEQ ID NO:170; aminoacid residues 16-181 of SEQ ID NO:171 which is encoded by nucleotides46-543 of SEQ ID NO:170; amino acid residues 16-180 of SEQ ID NO:171which is encoded by nucleotides 46-540 of SEQ ID NO:170; amino acidresidues 16-179 of SEQ ID NO:171 which is encoded by nucleotides 46-537of SEQ ID NO:170; amino acid residues 16-178 of SEQ ID NO:171 which isencoded by nucleotides 46-534 of SEQ ID NO:170; amino acid residues16-177 of SEQ ID NO:171 which is encoded by nucleotides 46-531 of SEQ IDNO:170; amino acid residues 16-176 of SEQ ID NO:171 which is encoded bynucleotides 46-528 of SEQ ID NO:170; amino acid residues 16-175 of SEQID NO:171 which is encoded by nucleotides 46-525 of SEQ ID NO:170; aminoacid residues 16-174 of SEQ ID NO:171 which is encoded by nucleotides46-522 of SEQ ID NO:170; amino acid residues 16-173 of SEQ ID NO:171which is encoded by nucleotides 46-519 of SEQ ID NO:170; and amino acidresidues 16-172 of SEQ ID NO:171 which is encoded by nucleotides 46-516of SEQ ID NO:170. The N-terminally and C-terminally modifiedbiologically active mutants of IL-29 C1 mutants of the present inventionmay also include an N-terminal Methione if expressed, for instance, inE. coli.

The IL-29 polypeptides of the present invention include, for example,SEQ ID NOs:4, 15, 27, 29, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93,95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 139, 141, 143,145, 147, 149, 151, 153, 155, 157, 159, 161, 171, 173 and 175, which areencoded by IL-29 polynucleotide molecules as shown in SEQ ID NOs:3, 14,26, 28, 40, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 138, 140, 142, 144, 146, 148,175, 152, 154, 156, 158, 160, 170, 172 and 174, respectively, C1 mutantsthereof, N-terminally modified C1 mutants thereof, C-terminally modifiedC1 mutants thereof, N-terminally and C-terminally C1 mutants thereof, C5mutants thereof, N-terminally modified C5 mutants thereof, C-terminallymodified C5 mutants thereof, N-terminally and C-terminally modified C5mutants thereof, fragments thereof, and fusion proteins thereof. TheIL-29 polypeptides may further include a signal sequence as shown in SEQID NO:119 or a signal sequence as shown in SEQ ID NO:121. Apolynucleotide molecule encoding the signal sequence polypeptide of SEQID NO:119 is shown as SEQ ID NO:118. A polynucleotide molecule encodingthe signal sequence polypeptide of SEQ ID NO:120 is shown as SEQ IDNO:121.

Table 3 sets forth the one-letter codes used within SEQ IDs 30, 31, 32,33, 34, and 35 to denote degenerate nucleotide positions. “Resolutions”are the nucleotides denoted by a code letter. “Complement” indicates thecode for the complementary nucleotide(s). For example, the code Ydenotes either C or T, and its complement R denotes A or G, with A beingcomplementary to T, and G being complementary to C.

TABLE 3 Nucleotide Resolution Complement Resolution A A T T C C G G G GC C T T A A R A|G Y C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|GW A|T W A|T H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T HA|C|T N A|C|G|T N A|C|G|T

The degenerate codons used in SEQ ID NOs: 30, 31, 32, 33, 34, and 35,encompassing all possible codons for a given amino acid, are set forthin Table 4.

TABLE 4 One Amino Letter Degenerate Acid Code Codons Codon Cys C TGC TGTTGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT ACN Pro PCCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA GGC GGG GGT GGNAsn N AAC AAT AAY Asp D GAC GAT GAY Glu E GAA GAG GAR Gln Q CAA CAG CARHis H CAC CAT CAY Arg R AGA AGG CGA CGC CGG CGT MGN Lys K AAA AAG AARMet M ATG ATG Ile I ATA ATC ATT ATH Leu L CTA CTC CTG CTT TTA TTG YTNVal V GTA GTC GTG GTT GTN Phe F TTC TTT TTY Tyr Y TAC TAT TAY Trp W TGGTGG Ter . TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any X NNN

One of ordinary skill in the art will appreciate that some ambiguity isintroduced in determining a degenerate codon, representative of allpossible codons encoding each amino acid. For example, the degeneratecodon for serine (WSN) can, in some circumstances, encode arginine(AGR), and the degenerate codon for arginine (MGN) can, in somecircumstances, encode serine (AGY). A similar relationship existsbetween codons encoding phenylalanine and leucine. Thus, somepolynucleotides encompassed by the degenerate sequence may encodevariant amino acid sequences, but one of ordinary skill in the art caneasily identify such variant sequences by reference to the amino acidsequence of SEQ ID NOS:19, 21, 23, 25, 27, and 29. Variant sequences canbe readily tested for functionality as described herein.

One of ordinary skill in the art will also appreciate that differentspecies can exhibit “preferential codon usage.” In general, see,Grantham, et al., Nuc. Acids Res. 8:1893-912, 1980; Haas, et al. Curr.Biol. 6:315-24, 1996; Wain-Hobson, et al., Gene 13:355-64, 1981;Grosjean and Fiers, Gene 18:199-209, 1982; Holm, Nuc. Acids Res.14:3075-87, 1986; Ikemura, J. Mol. Biol. 158:573-97, 1982. As usedherein, the term “preferential codon usage” or “preferential codons” isa term of art referring to protein translation codons that are mostfrequently used in cells of a certain species, thus favoring one or afew representatives of the possible codons encoding each amino acid (SeeTable 4). For example, the amino acid Threonine (Thr) may be encoded byACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonlyused codon; in other species, for example, insect cells, yeast, virusesor bacteria, different Thr codons may be preferential. Preferentialcodons for a particular species can be introduced into thepolynucleotides of the present invention by a variety of methods knownin the art. Introduction of preferential codon sequences intorecombinant DNA can, for example, enhance production of the protein bymaking protein translation more efficient within a particular cell typeor species. Therefore, the degenerate codon sequence disclosed in SEQ IDNOs: 30, 31, 32, 33, 34, and 35 serves as a template for optimizingexpression of polynucleotides in various cell types and species commonlyused in the art and disclosed herein. Sequences containing preferentialcodons can be tested and optimized for expression in various species,and tested for functionality as disclosed herein.

As previously noted, the isolated polynucleotides of the presentinvention include DNA and RNA. Methods for preparing DNA and RNA arewell known in the art. In general, RNA is isolated from a tissue or cellthat produces large amounts of IL-28 or IL-29 RNA. Such tissues andcells are identified by Northern blotting (Thomas, Proc. Natl. Acad.Sci. USA 77:5201, 1980), or by screening conditioned medium from variouscell types for activity on target cells or tissue. Once the activity orRNA producing cell or tissue is identified, total RNA can be preparedusing guanidinium isothiocyanate extraction followed by isolation bycentrifugation in a CsCl gradient (Chirgwin et al., Biochemistry18:52-94, 1979). Poly (A)±RNA is prepared from total RNA using themethod of Aviv and Leder (Proc. Natl. Acad. Sci. USA 69:1408-12, 1972).Complementary DNA (cDNA) is prepared from poly(A)⁺ RNA using knownmethods. In the alternative, genomic DNA can be isolated.Polynucleotides encoding IL-28 or IL-29 polypeptides are then identifiedand isolated by, for example, hybridization or PCR.

A full-length clones encoding IL-28 or IL-29 can be obtained byconventional cloning procedures. Complementary DNA (cDNA) clones arepreferred, although for some applications (e.g., expression intransgenic animals) it may be preferable to use a genomic clone, or tomodify a cDNA clone to include at least one genomic intron. Methods forpreparing cDNA and genomic clones are well known and within the level ofordinary skill in the art, and include the use of the sequence disclosedherein, or parts thereof, for probing or priming a library. Expressionlibraries can be probed with antibodies to IL-28 receptor fragments, orother specific binding partners.

Those skilled in the art will recognize that the sequence disclosed in,for example, SEQ ID NOs:1, 3, and 5, respectively, represent mutationsof single alleles of human IL-28 and IL-29 bands, and that allelicvariation and alternative splicing are expected to occur. For example,an IL-29 variant has been identified where amino acid residue 169 (Asn)as shown in SEQ ID NO:4 is an Arg residue, as described in WO 02/086087.Such allelic variants are included in the present invention. Allelicvariants of this sequence can be cloned by probing cDNA or genomiclibraries from different individuals according to standard procedures.Allelic variants of the DNA sequence shown in SEQ ID NOs:1, 3 and 5,including those containing silent mutations and those in which mutationsresult in amino acid sequence changes, in addition to the cysteinemutations, are within the scope of the present invention, as areproteins which are allelic variants of SEQ ID NOs:2, 4, and 6. cDNAsgenerated from alternatively spliced mRNAs, which retain the propertiesof IL-28 or IL-29 polypeptides, are included within the scope of thepresent invention, as are polypeptides encoded by such cDNAs and mRNAs.Allelic variants and splice variants of these sequences can be cloned byprobing cDNA or genomic libraries from different individuals or tissuesaccording to standard procedures known in the art, and mutations to thepolynucleotides encoding cysteines or cysteine residues can beintroduced as described herein.

Within embodiments of the invention, isolated IL-28- and IL-29-encodingnucleic acid molecules can hybridize under stringent conditions tonucleic acid molecules having the nucleotide sequence selected from thegroup of SEQ ID NOs:1, 3, 5, 12, 14, 16, 18, 20, 22, 24, 26, 28, 74, 76,78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108,110, 112, 114, 116, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140,142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168,170, 172 and 174 or to nucleic acid molecules having a nucleotidesequence complementary to SEQ ID NOs:1, 3, 5, 12, 14, 16, 18, 20, 22,24, 26, 28, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 122, 124, 126, 128, 130, 132,134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160,162, 164, 166, 168, 170, 172 and 174. In general, stringent conditionsare selected to be about 5° C. lower than the thermal melting point(T_(m)) for the specific sequence at a defined ionic strength and pH.The T_(m) is the temperature (under defined ionic strength and pH) atwhich 50% of the target sequence hybridizes to a perfectly matchedprobe.

A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA and DNA-RNA,can hybridize if the nucleotide sequences have some degree ofcomplementarity. Hybrids can tolerate mismatched base pairs in thedouble helix, but the stability of the hybrid is influenced by thedegree of mismatch. The T_(m) of the mismatched hybrid decreases by 1°C. for every 1-1.5% base pair mismatch. Varying the stringency of thehybridization conditions allows control over the degree of mismatch thatwill be present in the hybrid. The degree of stringency increases as thehybridization temperature increases and the ionic strength of thehybridization buffer decreases.

It is well within the abilities of one skilled in the art to adapt theseconditions for use with a particular polynucleotide hybrid. The T_(m)for a specific target sequence is the temperature (under definedconditions) at which 50% of the target sequence will hybridize to aperfectly matched probe sequence. Those conditions which influence theT_(m) include, the size and base pair content of the polynucleotideprobe, the ionic strength of the hybridization solution, and thepresence of destabilizing agents in the hybridization solution. Numerousequations for calculating T_(m) are known in the art, and are specificfor DNA, RNA and DNA-RNA hybrids and polynucleotide probe sequences ofvarying length (see, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition (Cold Spring Harbor Press 1989);Ausubel et al., (eds.), Current Protocols in Molecular Biology (JohnWiley and Sons, Inc. 1987); Berger and Kimmel (eds.), Guide to MolecularCloning Techniques, (Academic Press, Inc. 1987); and Wetmur, Crit. Rev.Biochem. Mol. Biol. 26:227 (1990)). Sequence analysis software such asOLIGO 6.0 (LSR; Long Lake, Minn.) and Primer Premier 4.0 (PremierBiosoft International; Palo Alto, Calif.), as well as sites on theInternet, are available tools for analyzing a given sequence andcalculating T_(m) based on user defined criteria. Such programs can alsoanalyze a given sequence under defined conditions and identify suitableprobe sequences. Typically, hybridization of longer polynucleotidesequences, >50 base pairs, is performed at temperatures of about 20-25°C. below the calculated T_(m). For smaller probes, <50 base pairs,hybridization is typically carried out at the T_(m) or 5-10° C. belowthe calculated T_(m). This allows for the maximum rate of hybridizationfor DNA-DNA and DNA-RNA hybrids.

Following hybridization, the nucleic acid molecules can be washed toremove non-hybridized nucleic acid molecules under stringent conditions,or under highly stringent conditions. Typical stringent washingconditions include washing in a solution of 0.5×-2×SSC with 0.1% sodiumdodecyl sulfate (SDS) at 55-65° C. That is, nucleic acid moleculesencoding a variant, cysteine mutant, or IL-28 or IL-29 polypeptideshybridize with a nucleic acid molecule having the nucleotide sequenceselected from the group of SEQ ID NOs:1, 3, 5, 12, 14, 16, 18, 20, 22,24, 26, 28, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 122, 124, 126, 128, 130, 132,134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158 160,162, 164, 166, 168, 170, 172 and 174 (or its complement) under stringentwashing conditions, in which the wash stringency is equivalent to0.5×-2×SSC with 0.1% SDS at 55-65° C., including 0.5×SSC with 0.1% SDSat 55° C., or 2×SSC with 0.1% SDS at 65° C. One of skill in the art canreadily devise equivalent conditions, for example, by substituting SSPEfor SSC in the wash solution.

Typical highly stringent washing conditions include washing in asolution of 0.1×-0.2×SSC with 0.1% sodium dodecyl sulfate (SDS) at50-65° C. In other words, nucleic acid molecules encoding a variant of aIL-28 or IL-29 polypeptide hybridize with a nucleic acid molecule havingthe nucleotide sequence selected from the group of SEQ ID NOs:1, 3, 5,12, 14, 16, 18, 20, 22, 24, 26, 28, 74, 76, 78, 80, 82, 84, 86, 88, 90,92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 122, 124,126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152,154, 156, 158, 160, 162, 164, 166, 168, 170, 172 and 174 (or itscomplement) under highly stringent washing conditions, in which the washstringency is equivalent to 0.1×-0.2×SSC with 0.1% SDS at 50-65° C.,including 0.1×SSC with 0.1% SDS at 50° C., or 0.2×SSC with 0.1% SDS at65° C.

The present invention also provides IL-28 or IL-29 polypeptides thathave a substantially similar sequence identity to the polypeptides ofthe present invention, for example SEQ ID NOs:2, 4, 6, 13, 15, 17, 19,21, 23, 25, 27, 29, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129,131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,159, 161, 163, 165, 167, 169, 171, 173 and 175. The term “substantiallysimilar sequence identity” is used herein to denote polypeptidescomprising at least 80%, at least 90%, at least 95%, or greater than95%, 96%, 97%, 98%, 99%, 99.5% sequence identity to the sequences shownin SEQ ID NOs: 2, 4, 6, 13, 15, 17, 19, 21, 23, 25, 27, 29, 41, 75, 77,79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141,143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169,171, 173 or 175 respectively, or their orthologs. The present inventionalso includes polypeptides that comprise an amino acid sequence havingat least 80%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 99.5%, or greater than 99.5% sequenceidentity to a polypeptide or fragment thereof of the present invention.The present invention further includes nucleic acid molecules thatencode such polypeptides. The IL-28 and IL-29 polypeptides of thepresent invention are preferably recombinant polypeptides. In anotheraspect, the IL-28 and IL-29 polypeptides of the present invention haveat least 15, at least 30, at least 45, or at least 60 sequential aminoacids. For example, an IL-29 polypeptide of the present inventionrelates to a polypeptide having at least 15, at least 30, at least 45,or at least 60 sequential amino acids from SEQ ID NOs:2, 4, 6, 13, 15,17, 19, 21, 23, 25, 27, 29, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93,95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127,129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155,157, 159, 161, 163, 165, 167, 169, 171, 173 or 175. Methods fordetermining percent identity are described below.

The present invention also contemplates variant nucleic acid moleculesthat can be identified using two criteria: a determination of thesimilarity between the encoded polypeptide with the amino acid sequenceof SEQ ID NOs:2, 4, 6, 13, 15, 17, 19, 21, 23, 25, 27, 29, 41, 75, 77,79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141,143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169,171, 173 or 175, and/or a hybridization assay, as described above. Suchvariants include nucleic acid molecules: (1) that hybridize with anucleic acid molecule having the nucleotide sequence of SEQ ID NOs:1, 3,5, 12, 14, 16, 18, 20, 22, 24, 26, 28, 74, 76, 78, 80, 82, 84, 86, 88,90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 122,124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150,152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172 or 174 (or itscomplement) under stringent washing conditions, in which the washstringency is equivalent to 0.5×-2×SSC with 0.1% SDS at 55-65° C.; or(2) that encode a polypeptide having at least 80%, at least 90%, atleast 95% or greater than 95%, 96%, 97%, 98%, 99% sequence identity tothe amino acid sequence of SEQ ID NOs:2, 4, 6, 13, 15, 17, 19, 21, 23,25, 27, 29, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133,135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161,163, 165, 167, 169, 171, 173 or 175. Alternatively, variants can becharacterized as nucleic acid molecules: (1) that hybridize with anucleic acid molecule having the nucleotide sequence of SEQ ID NOs:1, 3,5, 12, 14, 16, 18, 20, 22, 24, 26, 28, 74, 76, 78, 80, 82, 84, 86, 88,90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 122,124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150,152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172 or 174 (or itscomplement) under highly stringent washing conditions, in which the washstringency is equivalent to 0.1×-0.2×SSC with 0.1% SDS at 50-65° C.; and(2) that encode a polypeptide having at least 80%, at least 90%, atleast 95% or greater than 95%, 96%, 97%, 98%, 99% sequence identity tothe amino acid sequence of SEQ ID NOs:2, 4, 6, 13, 15, 17, 19, 21, 23,25, 27, 29, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133,135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161,162, 164, 166, 168, 170, 172 or 174.

The present invention further provides a polynucleotide encoding apolypeptide that treats, prevents, inhibits the progression of, delaythe onset of, and/or reduce the severity or inhibit at least one of theconditions or symptoms of a cancer as disclosed herein wherein theencoded polypeptide is a sequence selected from the group of SEQ IDNOs:2, 4, 6, 13, 15, 17, 19, 21, 23, 25, 27, 29, 41, 75, 77, 79, 81, 83,85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115,117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147,149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173 and 175.

Percent sequence identity is determined by conventional methods. See,for example, Altschul et al., Bull. Math. Bio. 48:603 (1986), andHenikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1992).Briefly, two amino acid sequences are aligned to optimize the alignmentscores using a gap opening penalty of 10, a gap extension penalty of 1,and the “BLOSUM62” scoring matrix of Henikoff and Henikoff (ibid.) asshown in Table 5 (amino acids are indicated by the standard one-lettercodes).

$\frac{{Total}\mspace{14mu} {number}\mspace{14mu} {of}\mspace{14mu} {identical}\mspace{14mu} {matches}}{\begin{bmatrix}{{length}\mspace{14mu} {of}\mspace{14mu} {the}\mspace{14mu} {longer}\mspace{14mu} {sequence}\mspace{14mu} {plus}\mspace{14mu} {the}} \\{{number}\mspace{14mu} {of}\mspace{14mu} {gaps}\mspace{14mu} {introduced}\mspace{14mu} {into}\mspace{14mu} {the}} \\{{longer}\mspace{14mu} {sequence}\mspace{14mu} {in}\mspace{14mu} {order}\mspace{14mu} {to}\mspace{14mu} {align}\mspace{14mu} {the}\mspace{14mu} {two}\mspace{14mu} {sequences}}\end{bmatrix}} \times 100$  

TABLE 5 A R N D C Q E G H I L K M F P S T W Y V A 4 R −1 5 N −2 0 6 D −2−2 1 6 C 0 −3 −3 −3 9 Q −1 1 0 0 −3 5 E −1 0 0 2 −4 2 5 G 0 −2 0 −1 −3−2 −2 6 H −2 0 1 −1 −3 0 0 −2 8 I −1 −3 −3 −3 −1 −3 −3 −4 −3 4 L −1 −2−3 −4 −1 −2 −3 −4 −3 2 4 K −1 2 0 −1 −3 1 1 −2 −1 −3 −2 5 M −1 −1 −2 −3−1 0 −2 −3 −2 1 2 −1 5 F −2 −3 −3 −3 −2 −3 −3 −3 −1 0 0 −3 0 6 P −1 −2−2 −1 −3 −1 −1 −2 −2 −3 −3 −1 −2 −4 7 S 1 −1 1 0 −1 0 0 0 −1 −2 −2 0 −1−2 −1 4 T 0 −1 0 −1 −1 −1 −1 −2 −2 −1 −1 −1 −1 −2 −1 1 5 W −3 −3 −4 −4−2 −2 −3 −2 −2 −3 −2 −3 −1 1 −4 −3 −2 11 Y −2 −2 −2 −3 −2 −1 −2 −3 2 −1−1 −2 −1 3 −3 −2 −2 2 7 V 0 −3 −3 −3 −1 −2 −2 −3 −3 3 1 −2 1 −1 −2 −2 0−3 −1 4

Those skilled in the art appreciate that there are many establishedalgorithms available to align two amino acid sequences. The “FASTA”similarity search algorithm of Pearson and Lipman is a suitable proteinalignment method for examining the level of identity shared by an aminoacid sequence disclosed herein and the amino acid sequence of a putativevariant IL-28 or IL-29. The FASTA algorithm is described by Pearson andLipman, Proc. Nat'l Acad. Sci. USA 85:2444 (1988), and by Pearson, Meth.Enzymol. 183:63 (1990).

Briefly, FASTA first characterizes sequence similarity by identifyingregions shared by the query sequence (e.g., SEQ ID NO:2) and a testsequence that have either the highest density of identities (if the ktupvariable is 1) or pairs of identities (if ktup=2), without consideringconservative amino acid substitutions, insertions, or deletions. The tenregions with the highest density of identities are then rescored bycomparing the similarity of all paired amino acids using an amino acidsubstitution matrix, and the ends of the regions are “trimmed” toinclude only those residues that contribute to the highest score. Ifthere are several regions with scores greater than the “cutoff” value(calculated by a predetermined formula based upon the length of thesequence and the ktup value), then the trimmed initial regions areexamined to determine whether the regions can be joined to form anapproximate alignment with gaps. Finally, the highest scoring regions ofthe two amino acid sequences are aligned using a modification of theNeedleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol. Biol.48:444 (1970); Sellers, SIAM J. Appl. Math. 26:787 (1974)), which allowsfor amino acid insertions and deletions. Preferred parameters for FASTAanalysis are: ktup=1, gap opening penalty=10, gap extension penalty=1,and substitution matrix=BLOSUM62. These parameters can be introducedinto a FASTA program by modifying the scoring matrix file (“SMATRIX”),as explained in Appendix 2 of Pearson, Meth. Enzymol. 183:63 (1990).

FASTA can also be used to determine the sequence identity of nucleicacid molecules using a ratio as disclosed above. For nucleotide sequencecomparisons, the ktup value can range between one to six, preferablyfrom three to six, most preferably three, with other parameters set asdefault.

Variant IL-28 or IL-29 polypeptides or polypeptides with substantiallysimilar sequence identity are characterized as having one or more aminoacid substitutions, deletions or additions. These changes are preferablyof a minor nature, that is conservative amino acid substitutions (seeTable 6) and other substitutions that do not significantly affect thefolding or activity of the polypeptide; small deletions, typically ofone to about 30 amino acids; and amino- or carboxyl-terminal extensions,such as an amino-terminal methionine residue, a small linker peptide ofup to about 20-25 residues, or an affinity tag. The present inventionthus includes polypeptides that comprise a sequence that is at least80%, preferably at least 90%, and more preferably at least 95%, at least96%, at least 97%, at least 98%, at least 99% or greater than 99%identical to the corresponding region of SEQ ID NOs: 2, 4, 6, 13, 15,17, 19, 21, 23, 25, 27, 29, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93,95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127,129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155,157, 159, 161, 163, 165, 167, 169, 171, 173 or 175. Polypeptidescomprising affinity tags can further comprise a proteolytic cleavagesite between the IL-28 and IL-29 polypeptide and the affinity tag.Preferred such sites include thrombin cleavage sites and factor Xacleavage sites.

TABLE 6 Conservative amino acid substitutions Basic: arginine lysinehistidine Acidic: glutamic acid aspartic acid Polar: glutamineasparagine Hydrophobic: leucine isoleucine valine Aromatic:phenylalanine tryptophan tyrosine Small: glycine alanine serinethreonine methionine

Determination of amino acid residues that comprise regions or domainsthat are critical to maintaining structural integrity can be determined.Within these regions one can determine specific residues that will bemore or less tolerant of change and maintain the overall tertiarystructure of the molecule. Methods for analyzing sequence structureinclude, but are not limited to alignment of multiple sequences withhigh amino acid or nucleotide identity, secondary structurepropensities, binary patterns, complementary packing and buried polarinteractions (Barton, Current Opin. Struct. Biol. 5:372-376, 1995 andCordes et al., Current Opin. Struct. Biol. 6:3-10, 1996). In general,when designing modifications to molecules or identifying specificfragments determination of structure will be accompanied by evaluatingactivity of modified molecules.

Amino acid sequence changes are made in IL-28 or IL-29 polypeptides soas to minimize disruption of higher order structure essential tobiological activity. For example, where the IL-28 or IL-29 polypeptidecomprises one or more helices, changes in amino acid residues will bemade so as not to disrupt the helix geometry and other components of themolecule where changes in conformation abate some critical function, forexample, binding of the molecule to its binding partners. The effects ofamino acid sequence changes can be predicted by, for example, computermodeling as disclosed above or determined by analysis of crystalstructure (see, e.g., Lapthorn et al., Nat. Struct. Biol. 2:266-268,1995). Other techniques that are well known in the art compare foldingof a variant protein to a standard molecule (e.g., the native protein).For example, comparison of the cysteine pattern in a variant andstandard molecules can be made. Mass spectrometry and chemicalmodification using reduction and alkylation provide methods fordetermining cysteine residues which are associated with disulfide bondsor are free of such associations (Bean et al., Anal. Biochem.201:216-226, 1992; Gray, Protein Sci. 2:1732-1748, 1993; and Pattersonet al., Anal. Chem. 66:3727-3732, 1994). It is generally believed thatif a modified molecule does not have the same cysteine pattern as thestandard molecule folding would be affected. Another well known andaccepted method for measuring folding is circular dichroism (CD).Measuring and comparing the CD spectra generated by a modified moleculeand standard molecule is routine (Johnson, Proteins 7:205-214, 1990).Crystallography is another well known method for analyzing folding andstructure. Nuclear magnetic resonance (NMR), digestive peptide mappingand epitope mapping are also known methods for analyzing folding andstructurally similarities between proteins and polypeptides (Schaanan etal., Science 257:961-964, 1992).

A Hopp/Woods hydrophilicity profile of the IL-28 or IL-29 polypeptidesequence as shown in SEQ ID NOs:2, 4, 6, 13, 15, 17, 19, 21, 23, 25, 27,29, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103,105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135,137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163,165, 167, 169, 171, 173 or 175 can be generated (Hopp et al., Proc.Natl. Acad. Sci. 78:3824-3828, 1981; Hopp, J. Immun. Meth. 88:1-18, 1986and Triquier et al., Protein Engineering 11:153-169, 1998). The profileis based on a sliding six-residue window. Buried G, S, and T residuesand exposed H, Y, and W residues were ignored. Those skilled in the artwill recognize that hydrophilicity or hydrophobicity will be taken intoaccount when designing modifications in the amino acid sequence of aIL-28 or IL-29 polypeptide, so as not to disrupt the overall structuraland biological profile. Of particular interest for replacement arehydrophobic residues selected from the group consisting of Val, Leu andIle or the group consisting of Met, Gly, Ser, Ala, Tyr and Trp.

The identities of essential amino acids can also be inferred fromanalysis of sequence similarity between IFN-α and members of the familyof IL-28A, IL-28B, and IL-29 (as shown in Tables 1 and 2). Using methodssuch as “FASTA” analysis described previously, regions of highsimilarity are identified within a family of proteins and used toanalyze amino acid sequence for conserved regions. An alternativeapproach to identifying a variant polynucleotide on the basis ofstructure is to determine whether a nucleic acid molecule encoding apotential variant IL-28 or IL-29 gene can hybridize to a nucleic acidmolecule as discussed above.

Other methods of identifying essential amino acids in the polypeptidesof the present invention are procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (Cunninghamand Wells, Science 244:1081 (1989), Bass et al., Proc. Natl. Acad. Sci.USA 88:4498 (1991), Coombs and Corey, “Site-Directed Mutagenesis andProtein Engineering,” in Proteins: Analysis and Design, Angeletti (ed.),pages 259-311 (Academic Press, Inc. 1998)). In the latter technique,single alanine mutations are introduced at every residue in themolecule, and the resultant molecules are tested for biological orbiochemical activity as disclosed below to identify amino acid residuesthat are critical to the activity of the molecule. See also, Hilton etal., J. Biol. Chem. 271:4699 (1996).

The present invention also includes functional fragments of IL-28 orIL-29 polypeptides and nucleic acid molecules encoding such functionalfragments. A “functional” IL-28 or IL-29 or fragment thereof as definedherein is characterized by its proliferative or differentiatingactivity, by its ability to induce or inhibit specialized cellfunctions, or by its ability to bind specifically to an anti-IL-28 orIL-29 antibody or IL-28 receptor (either soluble or immobilized). Thespecialized activities of IL-28 or IL-29 polypeptides and how to testfor them are disclosed herein. As previously described herein, IL-28 andIL-29 polypeptides are characterized by a six-helical-bundle. Thus, thepresent invention further provides fusion proteins encompassing: (a)polypeptide molecules comprising one or more of the helices describedabove; and (b) functional fragments comprising one or more of thesehelices. The other polypeptide portion of the fusion protein may becontributed by another helical-bundle cytokine or interferon, such asIFN-α, or by a non-native and/or an unrelated secretory signal peptidethat facilitates secretion of the fusion protein.

The IL-28 or IL-29 polypeptides of the present invention, includingfull-length polypeptides, cysteine mutant polypeptides, biologicallyactive fragments, and fusion polypeptides can be produced according toconventional techniques using cells into which have been introduced anexpression vector encoding the polypeptide. As used herein, “cells intowhich have been introduced an expression vector” include both cells thathave been directly manipulated by the introduction of exogenous DNAmolecules and progeny thereof that contain the introduced DNA. Suitablehost cells are those cell types that can be transformed or transfectedwith exogenous DNA and grown in culture, and include bacteria, fungalcells, and cultured higher eukaryotic cells. Techniques for manipulatingcloned DNA molecules and introducing exogenous DNA into a variety ofhost cells are disclosed by Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989, and Ausubel et al., eds., Current Protocolsin Molecular Biology, John Wiley and Sons, Inc., NY, 1987.

In general, a DNA sequence encoding a IL-28 or IL-29 polypeptide of thepresent invention is operably linked to other genetic elements requiredfor its expression, generally including a transcription promoter andterminator, within an expression vector. The vector will also commonlycontain one or more selectable markers and one or more origins ofreplication, although those skilled in the art will recognize thatwithin certain systems selectable markers may be provided on separatevectors, and replication of the exogenous DNA may be provided byintegration into the host cell genome. Selection of promoters,terminators, selectable markers, vectors and other elements is a matterof routine design within the level of ordinary skill in the art. Manysuch elements are described in the literature and are available throughcommercial suppliers.

To direct a IL-28 or IL-29 polypeptide into the secretory pathway of ahost cell, a secretory signal sequence (also known as a leader sequence,prepro sequence or pre sequence) is provided in the expression vector.The secretory signal sequence may be, for example, that of Cysteinemutant IL-28 or IL-29, e.g., SEQ ID NO:119 or SEQ ID NO:121, or may bederived from another secreted protein (e.g., t-PA; see, U.S. Pat. No.5,641,655) or synthesized de novo. The secretory signal sequence isoperably linked to the IL-28 or IL-29 DNA sequence, i.e., the twosequences are joined in the correct reading frame and positioned todirect the newly synthesized polypeptide into the secretory pathway ofthe host cell. Secretory signal sequences are commonly positioned 5′ tothe DNA sequence encoding the polypeptide of interest, although certainsignal sequences may be positioned elsewhere in the DNA sequence ofinterest (see, e.g., Welch et al., U.S. Pat. No. 5,037,743; Holland etal., U.S. Pat. No. 5,143,830).

A wide variety of suitable recombinant host cells includes, but is notlimited to, gram-negative prokaryotic host organisms. Suitable strainsof E. coli include W3110, K12-derived strains MM294, TG-1, JM-107, BL21,and UT5600. Other suitable strains include: BL21(DE3), BL21(DE3)pLysS,BL21(DE3)pLysE, DH1, DH4I, DH5, DH5I, DH51F′, DH51MCR, DH10B, DH10B/p3,DH11S, C600, HB101, JM101, JM105, JM109, JM110, K38, RR1, Y1088, Y1089,CSH18, ER1451, ER1647, E. coli K12, E. coli K12 RV308, E. coli K12 C600,E. coliHB101, E. coli K12 C600 R.sub.k-M.sub.k-, E. coli K12 RR1 (see,for example, Brown (ed.), Molecular Biology Labfax (Academic Press1991)). Other gram-negative prokaryotic hosts can include Serratia,Pseudomonas, Caulobacter. Prokaryotic hosts can include gram-positiveorganisms such as Bacillus, for example, B. subtilis and B.thuringienesis, and B. thuringienesis var. israelensis, as well asStreptomyces, for example, S. lividans, S. ambofaciens, S. fradiae, andS. griseofuscus. Suitable strains of Bacillus subtilus include BR151,YB886, MI119, MI120, and B170 (see, for example, Hardy, “BacillusCloning Methods,” in DNA Cloning: A Practical Approach, Glover (ed.)(IRL Press 1985)). Standard techniques for propagating vectors inprokaryotic hosts are well-known to those of skill in the art (see, forexample, Ausubel et al. (eds.), Short Protocols in Molecular Biology,3^(rd) Edition (John Wiley & Sons 1995); Wu et al., Methods in GeneBiotechnology (CRC Press, Inc. 1997)). In one embodiment, the methods ofthe present invention use IL-28 or IL-29 expressed in the W3110 strain,which has been deposited at the American Type Culture Collection (ATCC)as ATCC #27325.

When large scale production of IL-28 or IL-29 using the expressionsystem of the present invention is required, batch fermentation can beused. Generally, batch fermentation comprises that a first stage seedflask is prepared by growing E. coli strains expressing IL-28 or IL-29in a suitable medium in shake flask culture to allow for growth to anoptical density (OD) of between 5 and 20 at 600 nm. A suitable mediumwould contain nitrogen from a source(s) such as ammonium sulfate,ammonium phosphate, ammonium chloride, yeast extract, hydrolyzed animalproteins, hydrolyzed plant proteins or hydrolyzed caseins. Phosphatewill be supplied from potassium phosphate, ammonium phosphate,phosphoric acid or sodium phosphate. Other components would be magnesiumchloride or magnesium sulfate, ferrous sulfate or ferrous chloride, andother trace elements. Growth medium can be supplemented withcarbohydrates, such as fructose, glucose, galactose, lactose, andglycerol, to improve growth. Alternatively, a fed batch culture is usedto generate a high yield of IL-28 or IL-29 protein. The IL-28 or IL-29producing E. coli strains are grown under conditions similar to thosedescribed for the first stage vessel used to inoculate a batchfermentation.

Following fermentation the cells are harvested by centrifugation,re-suspended in homogenization buffer and homogenized, for example, inan APV-Gaulin homogenizer (Invensys APV, Tonawanda, New York) or othertype of cell disruption equipment, such as bead mills or sonicators.Alternatively, the cells are taken directly from the fermentor andhomogenized in an APV-Gaulin homogenizer. The washed inclusion body prepcan be solubilized using guanidine hydrochloride (5-8 M) or urea (7-8 M)containing a reducing agent such as beta mercaptoethanol (10-100 mM) ordithiothreitol (5-50 mM). The solutions can be prepared in Tris,phopshate, HEPES or other appropriate buffers. Inclusion bodies can alsobe solubilized with urea (2-4 M) containing sodium lauryl sulfate(0.1-2%). In the process for recovering purified IL-28 or IL-29 fromtransformed E. coli host strains in which the IL-28 or IL-29 isaccumulates as retractile inclusion bodies, the cells are disrupted andthe inclusion bodies are recovered by centrifugation. The inclusionbodies are then solubilized and denatured in 6 M guanidine hydrochloridecontaining a reducing agent. The reduced IL-28 or IL-29 is then oxidizedin a controlled renaturation step. Refolded IL-28 or IL-29 can be passedthrough a filter for clarification and removal of insoluble protein. Thesolution is then passed through a filter for clarification and removalof insoluble protein. After the IL-28 or IL-29 protein is refolded andconcentrated, the refolded IL-28 or IL-29 protein is captured in dilutebuffer on a cation exchange column and purified using hydrophobicinteraction chromatography.

Cultured mammalian cells are suitable hosts within the presentinvention. Methods for introducing exogenous DNA into mammalian hostcells include calcium phosphate-mediated transfection (Wigler et al.,Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603,1981: Graham and Van der Eb, Virology 52:456, 1973), electroporation(Neumann et al., EMBO J. 1:841-5, 1982), DEAE-dextran mediatedtransfection (Ausubel et al., ibid.), and liposome-mediated transfection(Hawley-Nelson et al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80,1993, and viral vectors (Miller and Rosman, BioTechniques 7:980-90,1989; Wang and Finer, Nature Med. 2:714-6, 1996). The production ofrecombinant polypeptides in cultured mammalian cells is disclosed, forexample, by Levinson et al., U.S. Pat. No. 4,713,339; Hagen et al., U.S.Pat. No. 4,784,950; Palmiter et al., U.S. Pat. No. 4,579,821; andRingold, U.S. Pat. No. 4,656,134. Suitable cultured mammalian cellsinclude the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK(ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No. CRL1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) and Chinese hamsterovary (e.g. CHO-K1; ATCC No. CCL 61) cell lines. Additional suitablecell lines are known in the art and available from public depositoriessuch as the American Type Culture Collection, Manassas, Va. In general,strong transcription promoters are preferred, such as promoters fromSV-40 or cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Othersuitable promoters include those from metallothionein genes (U.S. Pat.Nos. 4,579,821 and 4,601,978) and the adenovirus major late promoter.

Drug selection is generally used to select for cultured mammalian cellsinto which foreign DNA has been inserted. Such cells are commonlyreferred to as “transfectants”. Cells that have been cultured in thepresence of the selective agent and are able to pass the gene ofinterest to their progeny are referred to as “stable transfectants.” Apreferred selectable marker is a gene encoding resistance to theantibiotic neomycin. Selection is carried out in the presence of aneomycin-type drug, such as G-418 or the like. Selection systems canalso be used to increase the expression level of the gene of interest, aprocess referred to as “amplification.” Amplification is carried out byculturing transfectants in the presence of a low level of the selectiveagent and then increasing the amount of selective agent to select forcells that produce high levels of the products of the introduced genes.A preferred amplifiable selectable marker is dihydrofolate reductase,which confers resistance to methotrexate. Other drug resistance genes(e.g. hygromycin resistance, multi-drug resistance, puromycinacetyltransferase) can also be used. Alternative markers that introducean altered phenotype, such as green fluorescent protein, or cell surfaceproteins such as CD4, CD8, Class I MHC, placental alkaline phosphatasemay be used to sort transfected cells from untransfected cells by suchmeans as FACS sorting or magnetic bead separation technology.

Other higher eukaryotic cells can also be used as hosts, including plantcells, insect cells and avian cells. The use of Agrobacterium rhizogenesas a vector for expressing genes in plant cells has been reviewed bySinkar et al., J. Biosci. (Bangalore) 11:47-58, 1987. Transformation ofinsect cells and production of foreign polypeptides therein is disclosedby Guarino et al., U.S. Pat. No. 5,162,222 and WIPO publication WO94/06463. Insect cells can be infected with recombinant baculovirus,commonly derived from Autographa californica nuclear polyhedrosis virus(AcNPV). See, King, L. A. and Possee, R. D., The Baculovirus ExpressionSystem: A Laboratory Guide, London, Chapman & Hall; O'Reilly, D. R. etal., Baculovirus Expression Vectors: A Laboratory Manual, New York,Oxford University Press., 1994; and, Richardson, C. D., Ed., BaculovirusExpression Protocols. Methods in Molecular Biology, Totowa, N.J., HumanaPress, 1995. The second method of making recombinant baculovirusutilizes a transposon-based system described by Luckow (Luckow, V. A, etal., J Virol 67:4566-79, 1993). This system is sold in the Bac-to-Backit (Life Technologies, Rockville, Md.). This system utilizes a transfervector, pFastBac1™ (Life Technologies) containing a Tn7 transposon tomove the DNA encoding the IL-28 or IL-29 polypeptide into a baculovirusgenome maintained in E. coli as a large plasmid called a “bacmid.” ThepFastBac1™ transfer vector utilizes the AcNPV polyhedrin promoter todrive the expression of the gene of interest, in this case IL-28 orIL-29. However, pFastBac1™ can be modified to a considerable degree. Thepolyhedrin promoter can be removed and substituted with the baculovirusbasic protein promoter (also known as Pcor, p6.9 or MP promoter) whichis expressed earlier in the baculovirus infection, and has been shown tobe advantageous for expressing secreted proteins. See, Hill-Perkins, M.S. and Possee, R. D., J. Gen. Virol. 71:971-6, 1990; Bonning, B. C. etal., J. Gen. Virol. 75:1551-6, 1994; and, Chazenbalk, G. D., andRapoport, B., J. Biol. Chem. 270:1543-9, 1995. In such transfer vectorconstructs, a short or long version of the basic protein promoter can beused. Moreover, transfer vectors can be constructed which replace thenative IL-28 or IL-29 secretory signal sequences with secretory signalsequences derived from insect proteins. For example, a secretory signalsequence from Ecdysteroid Glucosyltransferase (EGT), honey bee Melittin(Invitrogen, Carlsbad, Calif.), or baculovirus gp67 (PharMingen, SanDiego, Calif.) can be used in constructs to replace the native IL-28 orIL-29 secretory signal sequence. In addition, transfer vectors caninclude an in-frame fusion with DNA encoding an epitope tag at the C- orN-terminus of the expressed IL-28 or IL-29 polypeptide, for example, aGlu-Glu epitope tag (Grussenmeyer, T. et al., Proc. Natl. Acad. Sci.82:7952-4, 1985). Using techniques known in the art, a transfer vectorcontaining IL-28 or IL-29 is transformed into E. coli, and screened forbacmids which contain an interrupted lacZ gene indicative of recombinantbaculovirus. The bacmid DNA containing the recombinant baculovirusgenome is isolated, using common techniques, and used to transfectSpodoptera frugiperda cells, e.g. Sf9 cells. Recombinant virus thatexpresses IL-28 or IL-29 is subsequently produced. Recombinant viralstocks are made by methods commonly used the art.

The recombinant virus is used to infect host cells, typically a cellline derived from the fall armyworm, Spodoptera frugiperda. See, ingeneral, Glick and Pasternak, Molecular Biotechnology: Principles andApplications of Recombinant DNA, ASM Press, Washington, D.C., 1994.Another suitable cell line is the High FiveO™ cell line (Invitrogen)derived from Trichoplusia ni (U.S. Pat. No. 5,300,435).

Fungal cells, including yeast cells, can also be used within the presentinvention. Yeast species of particular interest in this regard includeSaccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.Methods for transforming S. cerevisiae cells with exogenous DNA andproducing recombinant polypeptides therefrom are disclosed by, forexample, Kawasaki, U.S. Pat. No. 4,599,311; Kawasaki et al., U.S. Pat.No. 4,931,373; Brake, U.S. Pat. No. 4,870,008; Welch et al., U.S. Pat.No. 5,037,743; and Murray et al., U.S. Pat. No. 4,845,075. Transformedcells are selected by phenotype determined by the selectable marker,commonly drug resistance or the ability to grow in the absence of aparticular nutrient (e.g., leucine). A preferred vector system for usein Saccharomyces cerevisiae is the POT1 vector system disclosed byKawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformedcells to be selected by growth in glucose-containing media. Suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311; Kingsman etal., U.S. Pat. No. 4,615,974; and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446;5,063,154; 5,139,936 and 4,661,454. Transformation systems for otheryeasts, including Hansenula polymorpha, Schizosaccharomyces pombe,Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichiapastoris, Pichia methanolica, Pichia guillermondii and Candida maltosaare known in the art. See, for example, Gleeson et al., J. Gen.Microbiol. 132:3459-65, 1986 and Cregg, U.S. Pat. No. 4,882,279.Aspergillus cells may be utilized according to the methods of McKnightet al., U.S. Pat. No. 4,935,349. Methods for transforming Acremoniumchrysogenum are disclosed by Sumino et al., U.S. Pat. No. 5,162,228.Methods for transforming Neurospora are disclosed by Lambowitz, U.S.Pat. No. 4,486,533. The use of Pichia methanolica as host for theproduction of recombinant proteins is disclosed in U.S. Pat. Nos.5,955,349, 5,888,768 and 6,001,597, U.S. Pat. No. 5,965,389, U.S. Pat.No. 5,736,383, and U.S. Pat. No. 5,854,039.

It is preferred to purify the polypeptides and proteins of the presentinvention to 80% purity, more preferably to 90% purity, even morepreferably 95% purity, and particularly preferred is a pharmaceuticallypure state, that is greater than 99.9% pure with respect tocontaminating macromolecules, particularly other proteins and nucleicacids, and free of infectious and pyrogenic agents. Preferably, apurified polypeptide or protein is substantially free of otherpolypeptides or proteins, particularly those of animal origin.

Expressed recombinant IL-28 or IL-29 proteins (including chimericpolypeptides and multimeric proteins) are purified by conventionalprotein purification methods, typically by a combination ofchromatographic techniques. See, in general, Affinity Chromatography:Principles & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden,1988; and Scopes, Protein Purification: Principles and Practice,Springer-Verlag, New York, 1994. Proteins comprising a polyhistidineaffinity tag (typically about 6 histidine residues) are purified byaffinity chromatography on a nickel chelate resin. See, for example,Houchuli et al., Bio/Technol. 6: 1321-1325, 1988. Proteins comprising aglu-glu tag can be purified by immunoaffinity chromatography accordingto conventional procedures. See, for example, Grussenmeyer et al.,supra. Maltose binding protein fusions are purified on an amylose columnaccording to methods known in the art.

IL-28 or IL-29 polypeptides can also be prepared through chemicalsynthesis according to methods known in the art, including exclusivesolid phase synthesis, partial solid phase methods, fragmentcondensation or classical solution synthesis. See, for example,Merrifield, J. Am. Chem. Soc. 85:2149, 1963; Stewart et al., Solid PhasePeptide Synthesis (2nd edition), Pierce Chemical Co., Rockford, Ill.,1984; Bayer and Rapp, Chem. Pept. Prot. 3:3, 1986; and Atherton et al.,Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, Oxford,1989. In vitro synthesis is particularly advantageous for thepreparation of smaller polypeptides.

Using methods known in the art, IL-28 or IL-29 proteins can be preparedas monomers or multimers; glycosylated or non-glycosylated; pegylated ornon-pegylated; fusion proteins; and may or may not include an initialmethionine amino acid residue. IL-28 or IL-29 conjugates used fortherapy may comprise pharmaceutically acceptable water-soluble polymermoieties. Conjugation of interferons with water-soluble polymers hasbeen shown to enhance the circulating half-life of the interferon, andto reduce the immunogenicity of the polypeptide (see, for example,Nieforth et al., Clin. Pharmacol. Ther. 59:636 (1996), and Monkarsh etal., Anal. Biochem. 247:434 (1997)).

Suitable water-soluble polymers include polyethylene glycol (PEG),monomethoxy-PEG, mono-(C1-C10)alkoxy-PEG, aryloxy-PEG, poly-(N-vinylpyrrolidone)PEG, tresyl monomethoxy PEG, monomethoxy-PEGpropionaldehyde, PEG propionaldehyde, bis-succinimidyl carbonate PEG,propylene glycol homopolymers, a polypropylene oxide/ethylene oxideco-polymer, polyoxyethylated polyols (e.g., glycerol), monomethoxy-PEGbutyraldehyde, PEG butyraldehyde, monomethoxy-PEG acetaldehyde, PEGacetaldehyde, methoxyl PEG-succinimidyl propionate, methoxylPEG-succinimidyl butanoate, polyvinyl alcohol, dextran, cellulose, orother carbohydrate-based polymers. Suitable PEG may have a molecularweight from about 600 to about 60,000, including, for example, 5,000daltons, 12,000 daltons, 20,000 daltons, 30,000 daltons, and 40,000daltons, which can be linear or branched. A IL-28 or IL-29 conjugate canalso comprise a mixture of such water-soluble polymers.

One example of a IL-28 or IL-29 conjugate comprises a IL-28 or IL-29moiety and a polyalkyl oxide moiety attached to the N-terminus of theIL-28 or IL-29 moiety. PEG is one suitable polyalkyl oxide. As anillustration, IL-28 or IL-29 can be modified with PEG, a process knownas “PEGylation.” PEGylation of IL-28 or IL-29 can be carried out by anyof the PEGylation reactions known in the art (see, for example, EP 0 154316, Delgado et al., Critical Reviews in Therapeutic Drug CarrierSystems 9:249 (1992), Duncan and Spreafico, Clin. Pharmacokinet. 27:290(1994), and Francis et al., Int J Hematol 68:1 (1998)). For example,PEGylation can be performed by an acylation reaction or by an alkylationreaction with a reactive polyethylene glycol molecule. In an alternativeapproach, IL-28 or IL-29 conjugates are formed by condensing activatedPEG, in which a terminal hydroxy or amino group of PEG has been replacedby an activated linker (see, for example, Karasiewicz et al., U.S. Pat.No. 5,382,657).

PEGylation by acylation typically requires reacting an active esterderivative of PEG with a IL-28 or IL-29 polypeptide. An example of anactivated PEG ester is PEG esterified to N-hydroxysuccinimide As usedherein, the term “acylation” includes the following types of linkagesbetween IL-28 or IL-29 and a water-soluble polymer: amide, carbamate,urethane, and the like. Methods for preparing PEGylated IL-28 or IL-29by acylation will typically comprise the steps of (a) reacting an IL-28or IL-29 polypeptide with PEG (such as a reactive ester of an aldehydederivative of PEG) under conditions whereby one or more PEG groupsattach to IL-28 or IL-29, and (b) obtaining the reaction product(s).Generally, the optimal reaction conditions for acylation reactions willbe determined based upon known parameters and desired results. Forexample, the larger the ratio of PEG:IL-28 or IL-29, the greater thepercentage of polyPEGylated IL-28 or IL-29 product.

PEGylation by alkylation generally involves reacting a terminalaldehyde, e.g., propionaldehyde, butyraldehyde, acetaldehyde, and thelike, derivative of PEG with IL-28 or IL-29 in the presence of areducing agent. PEG groups are preferably attached to the polypeptidevia a —CH₂—NH₂ group.

Derivatization via reductive alkylation to produce a monoPEGylatedproduct takes advantage of the differential reactivity of differenttypes of primary amino groups available for derivatization. Typically,the reaction is performed at a pH that allows one to take advantage ofthe pKa differences between the c-amino groups of the lysine residuesand the α-amino group of the N-terminal residue of the protein. By suchselective derivatization, attachment of a water-soluble polymer thatcontains a reactive group such as an aldehyde, to a protein iscontrolled. The conjugation with the polymer occurs predominantly at theN-terminus of the protein without significant modification of otherreactive groups such as the lysine side chain amino groups.

Reductive alkylation to produce a substantially homogenous population ofmonopolymer IL-28 or IL-29 conjugate molecule can comprise the steps of:(a) reacting a IL-28 or IL-29 polypeptide with a reactive PEG underreductive alkylation conditions at a pH suitable to permit selectivemodification of the α-amino group at the amino terminus of the IL-28 orIL-29, and (b) obtaining the reaction product(s). The reducing agentused for reductive alkylation should be stable in aqueous solution andpreferably be able to reduce only the Schiff base formed in the initialprocess of reductive alkylation. Preferred reducing agents includesodium borohydride, sodium cyanoborohydride, dimethylamine borane,trimethylamine borane, and pyridine borane.

For a substantially homogenous population of monopolymer IL-28 or IL-29conjugates, the reductive alkylation reaction conditions are those thatpermit the selective attachment of the water-soluble polymer moiety tothe N-terminus of IL-28 or IL-29. Such reaction conditions generallyprovide for pKa differences between the lysine amino groups and theα-amino group at the N-terminus. The pH also affects the ratio ofpolymer to protein to be used. In general, if the pH is lower, a largerexcess of polymer to protein will be desired because the less reactivethe N-terminal α-group, the more polymer is needed to achieve optimalconditions. If the pH is higher, the polymer: IL-28 or IL-29 need not beas large because more reactive groups are available. Typically, the pHwill fall within the range of 3-9, or 3-6. Another factor to consider isthe molecular weight of the water-soluble polymer. Generally, the higherthe molecular weight of the polymer, the fewer number of polymermolecules which may be attached to the protein. For PEGylationreactions, the typical molecular weight is about 2 kDa to about 100 kDa,about 5 kDa to about 50 kDa, about 12 kDa to about 40 kDa, or about 20kDa to about 30 kDa. The molar ratio of water-soluble polymer to IL-28or IL-29 will generally be in the range of 1:1 to 100:1. Typically, themolar ratio of water-soluble polymer to IL-28 or IL-29 will be 1:1 to20:1 for polyPEGylation, and 1:1 to 5:1 for monoPEGylation.

General methods for producing conjugates comprising interferon andwater-soluble polymer moieties are known in the art. See, for example,Karasiewicz et al., U.S. Pat. No. 5,382,657, Greenwald et al., U.S. Pat.No. 5,738,846, Nieforth et al., Clin. Pharmacol. Ther. 59:636 (1996),Monkarsh et al., Anal. Biochem. 247:434 (1997). PEGylated species can beseparated from unconjugated IL-28 or IL-29 polypeptides using standardpurification methods, such as dialysis, ultrafiltration, ion exchangechromatography, affinity chromatography, size exclusion chromatography,and the like.

The IL-28 or IL-29 molecules of the present invention are capable ofspecifically binding the IL-28 receptor and/or acting as an antumoragent. The binding of IL-28 or 11-29 polypeptides to the IL-28 receptorcan be assayed using established approaches. IL-28 or IL-29 can beiodinated using an iodobead (Pierce, Rockford, Ill.) according tomanufacturer's directions, and the ¹²⁵I-IL-28 or ¹²⁵I-IL-29 can then beused as described below.

In a first approach fifty nanograms of ¹²⁵I-IL-28 or ¹²⁵I-IL-29 can becombined with 1000 ng of IL-28 receptor human IgG fusion protein, in thepresence or absence of possible binding competitors including unlabeledIL-28 or IL-29. The same binding reactions would also be performedsubstituting other cytokine receptor human IgG fusions as controls forspecificity. Following incubation at 4° C., protein-G (Zymed, SanFrancisco, Calif.) is added to the reaction, to capture the receptor-IgGfusions and any proteins bound to them, and the reactions are incubatedanother hour at 4° C. The protein-G sepharose is then collected, washedthree times with PBS and ¹²⁵I-IL-28 or ¹²⁵I-IL-29 bound is measure bygamma counter (Packard Instruments, Downers Grove, Ill.).

In a second approach, the ability of molecules to inhibit the binding of¹²⁵I-IL-28 or ¹²⁵I-IL-29 to plate bound receptors can be assayed. Afragment of the IL-28 receptor, representing the extracellular, ligandbinding domain, can be adsorbed to the wells of a 96 well plate byincubating 100 μl of 1 g/mL solution of receptor in the plate overnight.In a second form, a receptor-human IgG fusion can be bound to the wellsof a 96 well plate that has been coated with an antibody directedagainst the human IgG portion of the fusion protein. Following coatingof the plate with receptor the plate is washed, blocked with SUPERBLOCK(Pierce, Rockford, Ill.) and washed again. Solutions containing a fixedconcentration of ¹²⁵I-IL-28 or ¹²⁵I-IL-29 with or without increasingconcentrations of potential binding competitors including, IL-28, IL-29,IL-28 and IL-29, and 100 μl of the solution added to appropriate wellsof the plate. Following a one hour incubation at 4° C. the plate iswashed and the amount ¹²⁵I-IL-28 or ¹²⁵I-IL-29 bound determined bycounting (Topcount, Packard Instruments, Downers grove, IL). Thespecificity of binding of ¹²⁵I-IL-28 or ¹²⁵I-IL-29 can be defined byreceptor molecules used in these binding assays as well as by themolecules used as inhibitors.

In addition to pegylation, human albumin can be genetically coupled to apolypeptide of the present invention to prolong its half-life. Humanalbumin is the most prevalent naturally occurring blood protein in thehuman circulatory system, persisting in circulation in the body for overtwenty days. Research has shown that therapeutic proteins geneticallyfused to human albumin have longer half-lives. An IL28 or IL29 albuminfusion protein, like pegylation, may provide patients with long-actingtreatment options that offer a more convenient administration schedule,with similar or improved efficacy and safety compared to currentlyavailable treatments (U.S. Pat. No. 6,165,470; Syed et al., Blood,89(9):3243-3253 (1997); Yeh et al., Proc. Natl. Acad. Sci. USA,89:1904-1908 (1992); and Zeisel et al., Horm. Res., 37:5-13 (1992)).

Like the aforementioned peglyation and human albumin, an Fc portion ofthe human IgG molecule can be fused to a polypeptide of the presentinvention. The resultant fusion protein may have an increasedcirculating half-life due to the Fc moiety (U.S. Pat. No. 5,750,375,U.S. Pat. No. 5,843,725, U.S. Pat. No. 6,291,646; Barouch et al.,Journal of Immunology, 61:1875-1882 (1998); Barouch et al., Proc. Natl.Acad. Sci. USA, 97(8):4192-4197 (Apr. 11, 2000); and Kim et al.,Transplant Proc., 30(8):4031-4036 (December 1998)).

As used herein, the term “antibodies” includes polyclonal antibodies,monoclonal antibodies, antigen-binding fragments thereof such as F(ab′)₂and Fab fragments, single chain antibodies, and the like, includinggenetically engineered antibodies. Non-human antibodies may be humanizedby grafting non-human CDRs onto human framework and constant regions, orby incorporating the entire non-human variable domains (optionally“cloaking” them with a human-like surface by replacement of exposedresidues, wherein the result is a “veneered” antibody). In someinstances, humanized antibodies may retain non-human residues within thehuman variable region framework domains to enhance proper bindingcharacteristics. Through humanizing antibodies, biological half-life maybe increased, and the potential for adverse immune reactions uponadministration to humans is reduced. One skilled in the art can generatehumanized antibodies with specific and different constant domains (i.e.,different Ig subclasses) to facilitate or inhibit various immunefunctions associated with particular antibody constant domains.Antibodies are defined to be specifically binding if they bind to IL-28or IL-29 polypeptide or protein with an affinity at least 10-foldgreater than the binding affinity to control (non-IL-28 and IL-29)polypeptide or protein. The affinity of a monoclonal antibody can bereadily determined by one of ordinary skill in the art (see, forexample, Scatchard, Ann. NY Acad. Sci. 51: 660-672, 1949).

Methods for preparing polyclonal and monoclonal antibodies are wellknown in the art (see for example, Hurrell, J. G. R., Ed., MonoclonalHybridoma Antibodies: Techniques and Applications, CRC Press, Inc., BocaRaton, Fla., 1982, which is incorporated herein by reference). Thepolypeptide immunogen may be a full-length molecule or a portionthereof. If the polypeptide portion is “hapten-like”, such portion maybe advantageously joined or linked to a macromolecular carrier (such askeyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanustoxoid) for immunization.

A variety of assays known to those skilled in the art can be utilized todetect antibodies which specifically bind to IL-28 or IL-29polypeptides. Exemplary assays are described in detail in UsingAntibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold SpringHarbor Laboratory Press, 1999. Representative examples of such assaysinclude: concurrent immunoelectrophoresis, radio-immunoassays,radio-immunoprecipitations, enzyme-linked immunosorbent assays (ELISA),dot blot assays, Western blot assays, inhibition or competition assays,and sandwich assays.

For certain applications, including in vitro and in vivo diagnosticuses, it is advantageous to employ labeled antibodies. Suitable directtags or labels include radionuclides, enzymes, substrates, cofactors,inhibitors, fluorescent markers, chemiluminescent markers, magneticparticles and the like; indirect tags or labels may feature use ofbiotin-avidin or other complement/anti-complement pairs asintermediates. Antibodies of the present invention may also be directlyor indirectly conjugated to drugs, toxins, radionuclides and the like,and these conjugates used for in vivo diagnostic or therapeuticapplications (e.g., inhibition of cell proliferation). See, in general,Ramakrishnan et al., Cancer Res. 56:1324-1330, 1996.

Administration of a pharmaceutical formulation to a patient can betopical, inhalant, intravenous, intraarterial, intraperitoneal,intramuscular, subcutaneous, intrapleural, intrathecal, by perfusionthrough a regional catheter, or by direct intralesional injection. Whenadministering therapeutic proteins by injection, the administration maybe by continuous infusion or by single or multiple boluses. In general,pharmaceutical formulations will include a IL-28 or IL-29 polypeptide incombination with a pharmaceutically acceptable vehicle, such as saline,buffered saline, 5% dextrose in water, or the like. Formulations mayfurther include one or more excipients, preservatives, solubilizers,buffering agents, albumin to prevent protein loss on vial surfaces, etc.Methods of formulation are well known in the art and are disclosed, forexample, in Remington: The Science and Practice of Pharmacy, Gennaro,ed., Mack Publishing Co., Easton, Pa., 19^(th) ed., 1995. An IL-28 orIL-29 polypeptide will preferably be used in a concentration of about 10to 100 μg/ml of total volume, although concentrations in the range of 1ng/ml to 1000 μg/ml may be used. For topical application, such as forthe promotion of wound healing, the protein will be applied in the rangeof 0.1-10 μg/cm² of wound area, with the exact dose determined by theclinician according to accepted standards, taking into account thenature and severity of the condition to be treated, patient traits, etc.Determination of dose is within the level of ordinary skill in the art.Dosing is daily or intermittently over the period of treatment.Intravenous administration will be by bolus injection or infusion over atypical period of one to several hours. Sustained release formulationscan also be employed. In general, a therapeutically effective amount ofIL-28 or IL-29 is an amount sufficient to produce a clinicallysignificant change in the treated condition, such as a clinicallysignificant change in hematopoietic or immune function, a significantreduction in morbidity, or a significantly increased histological score.

As an illustration, pharmaceutical formulations may be supplied as a kitcomprising a container that comprises an IL-28 or IL29 polypeptide ofthe present invention. Therapeutic polypeptides can be provided in theform of an injectable solution for single or multiple doses, or as asterile powder that will be reconstituted before injection.Alternatively, such a kit can include a dry-powder disperser, liquidaerosol generator, or nebulizer for administration of a therapeuticpolypeptide. Such a kit may further comprise written information onindications and usage of the pharmaceutical composition. Moreover, suchinformation may include a statement that the IL-28 or IL29 polypeptideformulation is contraindicated in patients with known hypersensitivityto IL-28 or IL29 polypeptide.

B. The Use of IL-28 and IL-29 to Treat Cancer

IL-28 and IL-29 polypeptides of the present invention have been shown tohave an antiviral effect that is similar to interferon-α (See WO04/037995). Interferon has been approved in the United States fortreatment of autoimmune diseases, condyloma acuminatum, chronichepatitis C, bladder carcinoma, cervical carcinoma, laryngealpapillomatosis, fungoides mycosis, chronic hepatitis B, Kaposi's sarcomain patients infected with human immunodeficiency virus, malignantmelanoma, hairy cell leukemia, and multiple sclerosis. In addition,IL-28 and IL-29 polypeptides may be used to treat forms ofarteriosclerosis, such as atherosclerosis, by inhibiting cellproliferation. Accordingly, the present invention contemplates the useof IL-28 or IL-29 polypeptides, fusion proteins, and fragments thereofhaving IL-28 and IL-29 activity to treat such conditions, as well as totreat retinopathy.

The IL-28 polypeptides of the present invention encompass IL-28A andIL-28B polypeptides. IL-28A polypeptides of the present inventioninclude, for example, SEQ ID NOs:2, 13, 19, 21, 23, 25, 163 and 165which are encoded by IL-28A polynucleotide molecules as shown in SEQ IDNOs:1, 12, 18, 20, 22, 24, 162 and 164, respectively. In addition, thepresent invention also provides for IL-28A polypeptides as shown in SEQID NOs:36, 37, 38, and 39, C2 mutants thereof, N-terminally modified C2mutants thereof, C-terminally modified C2 mutants thereof, N-terminallyand C-terminally C2 mutants thereof, C3 mutants thereof, N-terminallymodified C3 mutants thereof, C-terminally modified C3 mutants thereof,N-terminally and C-terminally modified C3 mutants thereof, fragmentsthereof, and fusion proteins thereof. The IL-28B polypeptides of thepresent invention include, for example, SEQ ID NOs:6, 17, 123, 125, 127,129, 131, 133, 135, 137, 167 and 169, which are encoded by IL-28Bpolynucleotide molecules as shown in SEQ ID NOs:5, 16, 122, 124, 126,128, 130, 132, 134, 136, 166 and 168, respectively, C2 mutants thereof,N-terminally modified C2 mutants thereof, C-terminally modified C2mutants thereof, N-terminally and C-terminally C2 mutants thereof, C3mutants thereof, N-terminally modified C3 mutants thereof, C-terminallymodified C3 mutants thereof, N-terminally and C-terminally modified C3mutants thereof, fragments thereof, and fusion proteins thereof.

The IL-29 polypeptides of the present invention include, for example,SEQ ID NOs:4, 15, 27, 29, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93,95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 139, 141, 143,145, 147, 149, 151, 153, 155, 157, 159, 161, 171, 173 and 175, which areencoded by IL-29 polynucleotide molecules as shown in SEQ ID NOs:3, 14,26, 28, 40, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 138, 140, 142, 144, 146, 148,175, 152, 154, 156, 158, 160, 170, 172 and 174, respectively, C1 mutantsthereof, N-terminally modified C1 mutants thereof, C-terminally modifiedC1 mutants thereof, N-terminally and C-terminally C1 mutants thereof, C5mutants thereof, N-terminally modified C5 mutants thereof, C-terminallymodified C5 mutants thereof, N-terminally and C-terminally modified C5mutants thereof, fragments thereof, and fusion proteins thereof. TheIL-29 polypeptides may further include a signal sequence as shown in SEQID NO:119 or a signal sequence as shown in SEQ ID NO:121. Apolynucleotide molecule encoding the signal sequence polypeptide of SEQID NO:119 is shown as SEQ ID NO:118. A polynucleotide molecule encodingthe signal sequence polypeptide of SEQ ID NO:120 is shown as SEQ IDNO:121.

The present invention provides for the use of these IL-28 and IL-29proteins, polypeptides, and peptides having IL-28 and IL-29 activity totreat, prevent, inhibit the progression of, delay the onset of, and/orreduce at least one of the conditions or symptoms associated with thelymphoproliferative disorders, including for instance, B-cell lymphomas,chronic lymphocytic leukemia, acute lymphocytic leukemia, Non-Hodgkin'slymphomas, multiple myeloma, acute myelocytic leukemia, chronicmyelocytic leukemia. In addition, the present invention further providesfor the use of IL-28 and IL-29 proteins, polypeptides, and peptideshaving IL-28 and IL-29 activity to treat, prevent, inhibit theprogression of, delay the onset of, and/or reduce the severity orinhibit at least one of the conditions or symptoms associated with thefollowing cancers selected from the group of renal cell carcinoma,hepatocellular carcinoma, cervical cancer (e.g., squamous type andadenocarcinoma), head and neck tumours (e.g., Hypopharyngeal Cancer,Laryngeal Cancer, Lip and Oral Cavity Cancer, Metastatic Squamous NeckCancer with Occult Primary, Nasopharyngeal Cancer, Oropharyngeal Cancer,Paranasal Sinus and Nasal Cavity Cancer, Parathyroid Cancer, andSalivary Gland Cancer), melanoma (e.g., malignant melanoma such asSuperficial spreading melanoma, Nodular melanoma, and Lentigo malignamelanoma), thyroid carcinoma (e.g., Papillary, Follicular, Medullary,and Anaplastic), malignant gliomas (e.g., gliobastoma multiforme andanaplastic astrocytoma), breast cancer (e.g., ductal carcinoma), coloncancer, lung cancer (e.g., small cell lung cancer, non-small cell lungcancer such as Squamous cell carcinoma, Adenocarcinoma and Large cellcarcinoma, and mesothelioma), pancreatic cancer, prostate cancer,stomach cancer, ovarian cancer, testicular cancer, Kaposi's sarcoma, andbone cancer (e.g., Osteosarcoma, Ewing's sarcoma, Chondrosarcoma,Spindle cell sarcoma, and Chordoma).

Interferons have also been shown to induce the expression of antigens bycultured cells (see, for example, Auth et al., Hepatology 18:546 (1993),Guadagni et al., Int. J. Biol. Markers 9:53 (1994), Girolomoni et al.,Eur. J. Immunol. 25:2163 (1995), and Maciejewski et al., Blood 85:3183(1995). This activity enhances the ability to identify new tumorassociated antigens in vitro. Moreover, the ability of interferons toaugment the level of expression of human tumor antigens indicates thatinterferons can be useful in an adjuvant setting for immunotherapy orenhance immunoscintigraphy using anti-tumor antigen antibodies (Guadagniet al., Cancer Immunol. Immunother. 26:222 (1988); Guadagni et al., Int.J. Biol. Markers 9:53 (1994)). Thus, the present invention includes theuse of IL-28 or IL-29 proteins, polypeptides and peptides having IL-28and IL-29 activity as an adjuvant for immunotherapy or to improveimmunoscintigraphy using anti-tumor antigen antibodies.

The activity and effect of an IL-28 or IL-29 polypeptide on tumorprogression and metastasis can be measured in vivo. Several syngeneicmouse models have been developed to study the influence of polypeptides,compounds or other treatments on tumor progression. In these models,tumor cells passaged in culture are implanted into mice of the samestrain as the tumor donor. The cells will develop into tumors havingsimilar characteristics in the recipient mice, and metastasis will alsooccur in some of the models. Appropriate tumor models for our studiesinclude the Lewis lung carcinoma (ATCC No. CRL-1642) and B16 melanoma(ATCC No. CRL-6323), amongst others. These are both commonly used tumorlines, syngeneic to the C57BL6 mouse, that are readily cultured andmanipulated in vitro. Tumors resulting from implantation of either ofthese cell lines are capable of metastasis to the lung in C57BL6 mice.The Lewis lung carcinoma model has recently been used in mice toidentify an inhibitor of angiogenesis (O'Reilly M S, et al. Cell 79:315-328, 1994). C57BL6/J mice are treated with an experimental agenteither through daily injection of recombinant protein, agonist orantagonist or a one-time injection of recombinant adenovirus. Three daysfollowing this treatment, 10⁵ to 10⁶ cells are implanted under thedorsal skin. Alternatively, the cells themselves may be infected withrecombinant adenovirus, such as one expressing IL-28 and IL-29, beforeimplantation so that the protein is synthesized at the tumor site orintracellularly, rather than systemically. The mice normally developvisible tumors within 5 days. The tumors are allowed to grow for aperiod of up to 3 weeks, during which time they may reach a size of1500-1800 mm³ in the control treated group. Tumor size and body weightare carefully monitored throughout the experiment. At the time ofsacrifice, the tumor is removed and weighed along with the lungs and theliver. The lung weight has been shown to correlate well with metastatictumor burden. As an additional measure, lung surface metastases arecounted. The resected tumor, lungs and liver are prepared forhistopathological examination, immunohistochemistry, and in situhybridization, using methods known in the art and described herein. Theinfluence of the expressed polypeptide in question, e.g., Cysteinemutant IL-28 and IL-29, on the ability of the tumor to recruitvasculature and undergo metastasis can thus be assessed. In addition,aside from using adenovirus, the implanted cells can be transientlytransfected with IL-28 and IL-29. Use of stable IL-28 or IL-29transfectants as well as use of inducible promoters to activate IL-28 orIL-29 expression in vivo are known in the art and can be used in thissystem to assess induction of metastasis. Moreover, purified IL-28 orIL-29 conditioned media can be directly injected in to this mouse model,and hence be used in this system. For general reference see, O'Reilly MS, et al. Cell 79:315-328, 1994; and Rusciano D, et al. Murine Models ofLiver Metastasis. Invasion Metastasis 14:349-361, 1995.

The present invention provides for a method of treating cancercomprising administering to a patient in need thereof a therapeuticallyeffective amount of a polypeptide selected from the group of SEQ IDNOs:2, 4, 6, 13, 15, 17, 19, 21, 23, 25, 27, 29, 41, 75, 77, 79, 81, 83,85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115,117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147,149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173 and 175wherein the cancer is selected from the group of B-cell lymphomas,chronic lymphocytic leukemia, acute lymphocytic leukemia, Non-Hodgkin'slymphomas, multiple myeloma, acute myelocytic leukemia, chronicmyelocytic leukemia, renal cell carcinoma, hepatocellular carcinoma,cervical cancer, melanoma, thyroid carcinoma, malignant gliomas, breastcancer, colon cancer, lung cancer, pancreatic cancer, prostate cancer,stomach cancer, ovarian cancer, testicular cancer, Kaposi's sarcoma, andbone cancer. The polypeptide may further optionally include apolyethylene glycol moiety, which can be covalently linked to thepolypeptide (e.g., amino-terminally). The polyethylene glycol may belinear or branched. The polyethylene glycol may have a molecular weightof about 20 kD, 30 kD, or 40 kD. The polyethylene glycol may bemonomethoxy-PEG propionaldehyde. The patient upon which the polypeptideis administered may be a mammal, such as a human.

The present invention also provides a method of treating cancercomprising administering to a patient in need thereof a therapeuticallyeffective amount of a polypeptide having at least 90% or 95% sequenceidentity with a sequence selected from the group of SEQ ID NOs:2, 4, 6,13, 15, 17, 19, 21, 23, 25, 27, 29, 41, 75, 77, 79, 81, 83, 85, 87, 89,91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123,125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151,153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173 and 175, whereinthe cancer is selected from the group of B-cell lymphomas, chroniclymphocytic leukemia, acute lymphocytic leukemia, Non-Hodgkin'slymphomas, multiple myeloma, acute myelocytic leukemia, chronicmyelocytic leukemia, renal cell carcinoma, hepatocellular carcinoma,cervical cancer, melanoma, thyroid carcinoma, malignant gliomas, breastcancer, colon cancer, lung cancer, pancreatic cancer, prostate cancer,stomach cancer, ovarian cancer, testicular cancer, Kaposi's sarcoma, andbone cancer. The polypeptide may have at least 15, at least 30, at least45, or at least 60 sequential amino acids to SEQ ID NOs:2, 4, 6, 13, 15,17, 19, 21, 23, 25, 27, 29, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93,95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127,129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155,157, 159, 161, 163, 165, 167, 169, 171, 173 and 175. The polypeptide mayfurther optionally include a polyethylene glycol moiety, which can becovalently linked to the polypeptide (e.g., amino-terminally). Thepolyethylene glycol may be linear or branched. The polyethylene glycolmay have a molecular weight of about 20 kD, 30 kD, or 40 kD. Thepolyethylene glycol may be monomethoxy-PEG propionaldehyde. The patientupon which the polypeptide is administered may be a mammal, such as ahuman.

The present invention also provides a method of treating cancercomprising administering to a patient in need thereof a therapeuticallyeffective amount of a formulation comprising: a polypeptide having atleast 90% or 95% sequence identity with a sequence selected from thegroup of SEQ ID NOs:2, 4, 6, 13, 15, 17, 19, 21, 23, 25, 27, 29, 41, 75,77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141,143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169,171, 173 and 175; and a pharmaceutically acceptable vehicle; and whereinthe cancer is selected from the group of renal cell carcinoma,hepatocellular carcinoma, cervical cancer (e.g., squamous type andadenocarcinoma), head and neck tumours (e.g., Hypopharyngeal Cancer,Laryngeal Cancer, Lip and Oral Cavity Cancer, Metastatic Squamous NeckCancer with Occult Primary, Nasopharyngeal Cancer, Oropharyngeal Cancer,Paranasal Sinus and Nasal Cavity Cancer, Parathyroid Cancer, andSalivary Gland Cancer), melanoma (e.g., malignant melanoma such asSuperficial spreading melanoma, Nodular melanoma, and Lentigo malignamelanoma), thyroid carcinoma (e.g., Papillary, Follicular, Medullary,and Anaplastic), malignant gliomas (e.g., gliobastoma multiforme andanaplastic astrocytoma), breast cancer (e.g., ductal carcinoma), coloncancer, lung cancer, pancreatic cancer, prostate cancer, stomach cancer,ovarian cancer, testicular cancer, Kaposi's sarcoma, and bone cancer(e.g., Osteosarcoma, Ewing's sarcoma, Chondrosarcoma, Spindle cellsarcoma, and Chordoma). The polypeptide may have at least 15, at least30, at least 45, or at least 60 sequential amino acids to SEQ ID NOs:2,4, 6, 13, 15, 17, 19, 21, 23, 25, 27, 29, 41, 75, 77, 79, 81, 83, 85,87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149,151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173 and 175. Thepolypeptide may further optionally include a polyethylene glycol moiety,which can be covalently linked to the polypeptide (e.g.,amino-terminally). The polyethylene glycol may be linear or branched.The polyethylene glycol may have a molecular weight of about 20 kD, 30kD, or 40 kD. The polyethylene glycol may be monomethoxy-PEGpropionaldehyde. The patient upon which the polypeptide is administeredmay be a mammal, such as a human. The second polypeptide may be anInterferon molecule, such as Interferon-alpha, Interferon-beta, orInterferon-gamma, another therapeutic agent, such as IL-2 or IL-21, orcombination thereof.

The present invention also provides a method of treating cancercomprising administering to a patient in need thereof a therapeuticallyeffective amount of a formulation comprising: a polypeptide having atleast 90% or 95% sequence identity with a sequence selected from thegroup of SEQ ID NOs:2, 4, 6, 13, 15, 17, 19, 21, 23, 25, 27, 29, 41, 75,77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141,143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169,171, 173 and 175; a second polypeptide; a pharmaceutically acceptablevehicle; and wherein the cancer is selected from the group of renal cellcarcinoma, hepatocellular carcinoma, cervical cancer (e.g., squamoustype and adenocarcinoma), head and neck tumours (e.g., HypopharyngealCancer, Laryngeal Cancer, Lip and Oral Cavity Cancer, MetastaticSquamous Neck Cancer with Occult Primary, Nasopharyngeal Cancer,Oropharyngeal Cancer, Paranasal Sinus and Nasal Cavity Cancer,Parathyroid Cancer, and Salivary Gland Cancer), melanoma (e.g.,malignant melanoma such as Superficial spreading melanoma, Nodularmelanoma, and Lentigo maligna melanoma), thyroid carcinoma (e.g.,Papillary, Follicular, Medullary, and Anaplastic), malignant gliomas(e.g., gliobastoma multiforme and anaplastic astrocytoma), breast cancer(e.g., ductal carcinoma), colon cancer, lung cancer, pancreatic cancer,prostate cancer, stomach cancer, ovarian cancer, testicular cancer,Kaposi's sarcoma, and bone cancer (e.g., Osteosarcoma, Ewing's sarcoma,Chondrosarcoma, Spindle cell sarcoma, and Chordoma). The polypeptide mayhave at least 15, at least 30, at least 45, or at least 60 sequentialamino acids to SEQ ID NOs:2, 4, 6, 13, 15, 17, 19, 21, 23, 25, 27, 29,41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105,107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137,139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165,167, 169, 171, 173 and 175. The polypeptide may further optionallyinclude a polyethylene glycol moiety, which can be covalently linked tothe polypeptide (e.g., amino-terminally). The polyethylene glycol may belinear or branched. The polyethylene glycol may have a molecular weightof about 20 kD, 30 kD, or 40 kD. The polyethylene glycol may bemonomethoxy-PEG propionaldehyde. The patient upon which the polypeptideis administered may be a mammal, such as a human. The second polypeptidemay be an Interferon molecule, such as Interferon-alpha,Interferon-beta, or Interferon-gamma, another therapeutic agent, such asIL-2 or IL-21, or combination thereof.

The present invention also provides a method of inhibiting theprogressive of cancer comprising administering to a patient in needthereof a therapeutically effective amount of a polypeptide having atleast 90% or 95% sequence identity with a sequence selected from thegroup of SEQ ID NOs:2, 4, 6, 13, 15, 17, 19, 21, 23, 25, 27, 29, 41, 75,77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141,143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169,171, 173 and 175, wherein the cancer is selected from the group ofB-cell lymphomas, chronic lymphocytic leukemia, acute lymphocyticleukemia, Non-Hodgkin's lymphomas, multiple myeloma, acute myelocyticleukemia, chronic myelocytic leukemia, renal cell carcinoma,hepatocellular carcinoma, cervical cancer, melanoma, thyroid carcinoma,malignant gliomas, breast cancer, colon cancer, lung cancer, pancreaticcancer, prostate cancer, stomach cancer, ovarian cancer, testicularcancer, Kaposi's sarcoma, and bone cancer. The polypeptide may have atleast 15, at least 30, at least 45, or at least 60 sequential aminoacids to SEQ ID NOs:2, 4, 6, 13, 15, 17, 19, 21, 23, 25, 27, 29, 41, 75,77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141,143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169,171, 173 and 175. The polypeptide may further optionally include apolyethylene glycol moiety, which can be covalently linked to thepolypeptide (e.g., amino-terminally). The polyethylene glycol may belinear or branched. The polyethylene glycol may have a molecular weightof about 20 kD, 30 kD, or 40 kD. The polyethylene glycol may bemonomethoxy-PEG propionaldehyde. The patient upon which the polypeptideis administered may be a mammal, such as a human.

The present invention also provides a method of inhibiting theprogression of cancer comprising administering to a patient in needthereof a therapeutically effective amount of a formulation comprising:a polypeptide having at least 90% or 95% sequence identity with asequence selected from the group of SEQ ID NOs:2, 4, 6, 13, 15, 17, 19,21, 23, 25, 27, 29, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129,131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,159, 161, 163, 165, 167, 169, 171, 173 and 175; a second polypeptide; apharmaceutically acceptable vehicle; and wherein the cancer is selectedfrom the group of renal cell carcinoma, hepatocellular carcinoma,cervical cancer (e.g., squamous type and adenocarcinoma), head and necktumours (e.g., Hypopharyngeal Cancer, Laryngeal Cancer, Lip and OralCavity Cancer, Metastatic Squamous Neck Cancer with Occult Primary,Nasopharyngeal Cancer, Oropharyngeal Cancer, Paranasal Sinus and NasalCavity Cancer, Parathyroid Cancer, and Salivary Gland Cancer), melanoma(e.g., malignant melanoma such as Superficial spreading melanoma,Nodular melanoma, and Lentigo maligna melanoma), thyroid carcinoma(e.g., Papillary, Follicular, Medullary, and Anaplastic), malignantgliomas (e.g., gliobastoma multiforme and anaplastic astrocytoma),breast cancer (e.g., ductal carcinoma), colon cancer, lung cancer,pancreatic cancer, prostate cancer, stomach cancer, ovarian cancer,testicular cancer, Kaposi's sarcoma, and bone cancer (e.g.,Osteosarcoma, Ewing's sarcoma, Chondrosarcoma, Spindle cell sarcoma, andChordoma). The polypeptide may have at least 15, at least 30, at least45, or at least 60 sequential amino acids to SEQ ID NOs:2, 4, 6, 13, 15,17, 19, 21, 23, 25, 27, 29, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93,95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127,129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155,157, 159, 161, 163, 165, 167, 169, 171, 173 and 175. The polypeptide mayfurther optionally include a polyethylene glycol moiety, which can becovalently linked to the polypeptide (e.g., amino-terminally). Thepolyethylene glycol may be linear or branched. The polyethylene glycolmay have a molecular weight of about 20 kD, 30 kD, or 40 kD. Thepolyethylene glycol may be monomethoxy-PEG propionaldehyde. The patientupon which the polypeptide is administered may be a mammal, such as ahuman. The second polypeptide may be an Interferon molecule, such asInterferon-alpha, Interferon-beta, or Interferon-gamma, anothertherapeutic agent, such as IL-2 or IL-21, or combination thereof.

The present invention also provides a method of delaying the onset ofcancer comprising administering to a patient in need thereof atherapeutically effective amount of a polypeptide having at least 90% or95% sequence identity with a sequence selected from the group of SEQ IDNOs:2, 4, 6, 13, 15, 17, 19, 21, 23, 25, 27, 29, 41, 75, 77, 79, 81, 83,85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115,117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147,149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173 and 175,wherein the cancer is selected from the group of B-cell lymphomas,chronic lymphocytic leukemia, acute lymphocytic leukemia, Non-Hodgkin'slymphomas, multiple myeloma, acute myelocytic leukemia, chronicmyelocytic leukemia, renal cell carcinoma, hepatocellular carcinoma,cervical cancer, melanoma, thyroid carcinoma, malignant gliomas, breastcancer, colon cancer, lung, cancer, pancreatic cancer, prostate cancer,stomach cancer, ovarian cancer, testicular cancer, Kaposi's sarcoma, andbone cancer. The polypeptide may have at least 15, at least 30, at least45, or at least 60 sequential amino acids to SEQ ID NOs:2, 4, 6, 13, 15,17, 19, 21, 23, 25, 27, 29, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93,95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127,129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155,157, 159, 161, 163, 165, 167, 169, 171, 173 and 175. The polypeptide mayfurther optionally include a polyethylene glycol moiety, which can becovalently linked to the polypeptide (e.g., amino-terminally). Thepolyethylene glycol may be linear or branched. The polyethylene glycolmay have a molecular weight of about 20 kD, 30 kD, or 40 kD. Thepolyethylene glycol may be monomethoxy-PEG propionaldehyde. The patientupon which the polypeptide is administered may be a mammal, such as ahuman.

The present invention also provides a method of delaying the onset ofcancer comprising administering to a patient in need thereof atherapeutically effective amount of a formulation comprising: apolypeptide having at least 90% or 95% sequence identity with a sequenceselected from the group of SEQ ID NOs:2, 4, 6, 13, 15, 17, 19, 21, 23,25, 27, 29, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133,135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161,163, 165, 167, 169, 171, 173 and 175; a second polypeptide; apharmaceutically acceptable vehicle; and wherein the cancer is selectedfrom the group of renal cell carcinoma, hepatocellular carcinoma,cervical cancer (e.g., squamous type and adenocarcinoma), head and necktumours (e.g., Hypopharyngeal Cancer, Laryngeal Cancer, Lip and OralCavity Cancer, Metastatic Squamous Neck Cancer with Occult Primary,Nasopharyngeal Cancer, Oropharyngeal Cancer, Paranasal Sinus and NasalCavity Cancer, Parathyroid Cancer, and Salivary Gland Cancer), melanoma(e.g., malignant melanoma such as Superficial spreading melanoma,Nodular melanoma, and Lentigo maligna melanoma), thyroid carcinoma(e.g., Papillary, Follicular, Medullary, and Anaplastic), malignantgliomas (e.g., gliobastoma multiforme and anaplastic astrocytoma),breast cancer (e.g., ductal carcinoma), colon cancer, lung cancer,pancreatic cancer, prostate cancer, stomach cancer, ovarian cancer,testicular cancer, Kaposi's sarcoma, and bone cancer (e.g.,Osteosarcoma, Ewing's sarcoma, Chondrosarcoma, Spindle cell sarcoma, andChordoma). The polypeptide may have at least 15, at least 30, at least45, or at least 60 sequential amino acids to SEQ ID NOs:2, 4, 6, 13, 15,17, 19, 21, 23, 25, 27, 29, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93,95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127,129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155,157, 159, 161, 163, 165, 167, 169, 171, 173 and 175. The polypeptide mayfurther optionally include a polyethylene glycol moiety, which can becovalently linked to the polypeptide (e.g., amino-terminally). Thepolyethylene glycol may be linear or branched. The polyethylene glycolmay have a molecular weight of about 20 kD, 30 kD, or 40 kD. Thepolyethylene glycol may be monomethoxy-PEG propionaldehyde. The patientupon which the polypeptide is administered may be a mammal, such as ahuman. The second polypeptide may be an Interferon molecule, such asInterferon-alpha, Interferon-beta, or Interferon-gamma, anothertherapeutic agent, such as IL-2 or IL-21, or combination thereof.

The present invention also provides a method of reducing the severity ofcancer comprising administering to a patient in need thereof atherapeutically effective amount of a polypeptide having at least 90% or95% sequence identity with a sequence selected from the group of SEQ IDNOs:2, 4, 6, 13, 15, 17, 19, 21, 23, 25, 27, 29, 41, 75, 77, 79, 81, 83,85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115,117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147,149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173 and 175,wherein the cancer is selected from the group of B-cell lymphomas,chronic lymphocytic leukemia, acute lymphocytic leukemia, Non-Hodgkin'slymphomas, multiple myeloma, acute myelocytic leukemia, chronicmyelocytic leukemia, renal cell carcinoma, hepatocellular carcinoma,cervical cancer, melanoma, thyroid carcinoma, malignant gliomas, breastcancer, colon cancer, lung cancer, pancreatic cancer, prostate cancer,stomach cancer, ovarian cancer, testicular cancer, Kaposi's sarcoma, andbone cancer. The polypeptide may have at least 15, at least 30, at least45, or at least 60 sequential amino acids to SEQ ID NOs:2, 4, 6, 13, 15,17, 19, 21, 23, 25, 27, 29, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93,95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127,129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155,157, 159, 161, 163, 165, 167, 169, 171, 173 and 175. The polypeptide mayfurther optionally include a polyethylene glycol moiety, which can becovalently linked to the polypeptide (e.g., amino-terminally). Thepolyethylene glycol may be linear or branched. The polyethylene glycolmay have a molecular weight of about 20 kD, 30 kD, or 40 kD. Thepolyethylene glycol may be monomethoxy-PEG propionaldehyde. The patientupon which the polypeptide is administered may be a mammal, such as ahuman.

The present invention also provides a method of reducing the severity ofcancer comprising administering to a patient in need thereof atherapeutically effective amount of a formulation comprising: apolypeptide having at least 90% or 95% sequence identity with a sequenceselected from the group of SEQ ID NOs:2, 4, 6, 13, 15, 17, 19, 21, 23,25, 27, 29, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133,135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161,163, 165, 167, 169, 171, 173 and 175; a second polypeptide; apharmaceutically acceptable vehicle; and wherein the cancer is selectedfrom the group of renal cell carcinoma, hepatocellular carcinoma,cervical cancer (e.g., squamous type and adenocarcinoma), head and necktumours (e.g., Hypopharyngeal Cancer, Laryngeal Cancer, Lip and OralCavity Cancer, Metastatic Squamous Neck Cancer with Occult Primary,Nasopharyngeal Cancer, Oropharyngeal Cancer, Paranasal Sinus and NasalCavity Cancer, Parathyroid Cancer, and Salivary Gland Cancer), melanoma(e.g., malignant melanoma such as Superficial spreading melanoma,Nodular melanoma, and Lentigo maligna melanoma), thyroid carcinoma(e.g., Papillary, Follicular, Medullary, and Anaplastic), malignantgliomas (e.g., gliobastoma multiforme and anaplastic astrocytoma),breast cancer (e.g., ductal carcinoma), colon cancer, lung cancer,pancreatic cancer, prostate cancer, stomach cancer, ovarian cancer,testicular cancer, Kaposi's sarcoma, and bone cancer (e.g.,Osteosarcoma, Ewing's sarcoma, Chondrosarcoma, Spindle cell sarcoma, andChordoma). The polypeptide may have at least 15, at least 30, at least45, or at least 60 sequential amino acids to SEQ ID NOs:2, 4, 6, 13, 15,17, 19, 21, 23, 25, 27, 29, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93,95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127,129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155,157, 159, 161, 163, 165, 167, 169, 171, 173 and 175. The polypeptide mayfurther optionally include a polyethylene glycol moiety, which can becovalently linked to the polypeptide (e.g., amino-terminally). Thepolyethylene glycol may be linear or branched. The polyethylene glycolmay have a molecular weight of about 20 kD, 30 kD, or 40 kD. Thepolyethylene glycol may be monomethoxy-PEG propionaldehyde. The patientupon which the polypeptide is administered may be a mammal, such as ahuman. The second polypeptide may be an Interferon molecule, such asInterferon-alpha, Interferon-beta, or Interferon-gamma, anothertherapeutic agent, such as IL-2 or IL-21, or combination thereof.

The present invention also provides a method of inhibiting at least oneof the conditions or symptoms of cancer comprising administering to apatient in need thereof a therapeutically effective amount of apolypeptide having at least 90% or 95% sequence identity with a sequenceselected from the group of SEQ ID NOs:2, 4, 6, 13, 15, 17, 19, 21, 23,25, 27, 29, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133,135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161,163, 165, 167, 169, 171, 173 and 175, wherein the cancer is selectedfrom the group of B-cell lymphomas, chronic lymphocytic leukemia, acutelymphocytic leukemia, Non-Hodgkin's lymphomas, multiple myeloma, acutemyelocytic leukemia, chronic myelocytic leukemia, renal cell carcinoma,hepatocellular carcinoma, cervical cancer, melanoma, thyroid carcinoma,malignant gliomas, breast cancer, colon cancer, lung cancer, pancreaticcancer, prostate cancer, stomach cancer, ovarian cancer, testicularcancer, Kaposi's sarcoma, and bone cancer. The polypeptide may have atleast 15, at least 30, at least 45, or at least 60 sequential aminoacids to SEQ ID NOs:2, 4, 6, 13, 15, 17, 19, 21, 23, 25, 27, 29, 41, 75,77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141,143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169,171, 173 and 175. The polypeptide may further optionally include apolyethylene glycol moiety, which can be covalently linked to thepolypeptide (e.g., amino-terminally). The polyethylene glycol may belinear or branched. The polyethylene glycol may have a molecular weightof about 20 kD, 30 kD, or 40 kD. The polyethylene glycol may bemonomethoxy-PEG propionaldehyde. The patient upon which the polypeptideis administered may be a mammal, such as a human.

The present invention also provides a method of inhibiting at least oneof the conditions or symptoms of cancer comprising administering to apatient in need thereof a therapeutically effective amount of aformulation comprising: a polypeptide having at least 90% or 95%sequence identity with a sequence selected from the group of SEQ IDNOs:2, 4, 6, 13, 15, 17, 19, 21, 23, 25, 27, 29, 41, 75, 77, 79, 81, 83,85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115,117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147,149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173 and 175;a second polypeptide; a pharmaceutically acceptable vehicle; and whereinthe cancer is selected from the group of renal cell carcinoma,hepatocellular carcinoma, cervical cancer (e.g., squamous type andadenocarcinoma), head and neck tumours (e.g., Hypopharyngeal Cancer,Laryngeal Cancer, Lip and Oral Cavity Cancer, Metastatic Squamous NeckCancer with Occult Primary, Nasopharyngeal Cancer, Oropharyngeal Cancer,Paranasal Sinus and Nasal Cavity Cancer, Parathyroid Cancer, andSalivary Gland Cancer), melanoma (e.g., malignant melanoma such asSuperficial spreading melanoma, Nodular melanoma, and Lentigo malignamelanoma), thyroid carcinoma (e.g., Papillary, Follicular, Medullary,and Anaplastic), malignant gliomas (e.g., gliobastoma multiforme andanaplastic astrocytoma), breast cancer (e.g., ductal carcinoma), coloncancer, lung cancer, pancreatic cancer, prostate cancer, stomach cancer,ovarian cancer, testicular cancer, Kaposi's sarcoma, and bone cancer(e.g., Osteosarcoma, Ewing's sarcoma, Chondrosarcoma, Spindle cellsarcoma, and Chordoma). The polypeptide may have at least 15, at least30, at least 45, or at least 60 sequential amino acids to SEQ ID NOs:2,4, 6, 13, 15, 17, 19, 21, 23, 25, 27, 29, 41, 75, 77, 79, 81, 83, 85,87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149,151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173 and 175. Thepolypeptide may further optionally include a polyethylene glycol moiety,which can be covalently linked to the polypeptide (e.g.,amino-terminally). The polyethylene glycol may be linear or branched.The polyethylene glycol may have a molecular weight of about 20 kD, 30kD, or 40 kD. The polyethylene glycol may be monomethoxy-PEGpropionaldehyde. The patient upon which the polypeptide is administeredmay be a mammal, such as a human. The second polypeptide may be anInterferon molecule, such as Interferon-alpha, Interferon-beta, orInterferon-gamma, another therapeutic agent, such as IL-2 or IL-21, orcombination thereof.

The present invention also provides a method of inhibiting at least oneof the conditions or symptoms of non-Hogkin's lymphoma comprisingadministering to a patient in need thereof a therapeutically effectiveamount of a polypeptide having at least 90% or 95% sequence identitywith a sequence selected from the group of SEQ ID NOs:2, 4, 6, 13, 15,17, 19, 21, 23, 25, 27, 29, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93,95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127,129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155,157, 159, 161, 163, 165, 167, 169, 171, 173 and 175, wherein the atleast one of the conditions or symptoms is selected from the group ofpainless swelling of a lymph node in the neck, armpit or groin, nightsweats, unexplained fever, weight loss, and excessive tiredness. Thepolypeptide may have at least 15, at least 30, at least 45, or at least60 sequential amino acids to SEQ ID NOs:2, 4, 6, 13, 15, 17, 19, 21, 23,25, 27, 29, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133,135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161,163, 165, 167, 169, 171, 173 and 175. The polypeptide may furtheroptionally include a polyethylene glycol moiety, which can be covalentlylinked to the polypeptide (e.g., amino-terminally). The polyethyleneglycol may be linear or branched. The polyethylene glycol may have amolecular weight of about 20 kD, 30 kD, or 40 kD. The polyethyleneglycol may be monomethoxy-PEG propionaldehyde. The patient upon whichthe polypeptide is administered may be a mammal, such as a human.

The present invention also provides a method of inhibiting at least oneof the conditions or symptoms of non-Hodgkin's lymphoma comprisingadministering to a patient in need thereof a therapeutically effectiveamount of a formulation comprising: a polypeptide having at least 90% or95% sequence identity with a sequence selected from the group of SEQ IDNOs:2, 4, 6, 13, 15, 17, 19, 21, 23, 25, 27, 29, 41, 75, 77, 79, 81, 83,85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115,117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147,149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173 and 175;a second polypeptide; and a pharmaceutically acceptable vehicle; whereinthe at least one of the conditions or symptoms is selected from thegroup of painless swelling of a lymph node in the neck, armpit or groin,night sweats, unexplained fever, weight loss, and excessive tiredness.The polypeptide may have at least 15, at least 30, at least 45, or atleast 60 sequential amino acids to SEQ ID NOs:2, 4, 6, 13, 15, 17, 19,21, 23, 25, 27, 29, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129,131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,159, 161, 163, 165, 167, 169, 171, 173 and 175. The polypeptide mayfurther optionally include a polyethylene glycol moiety, which can becovalently linked to the polypeptide (e.g., amino-terminally). Thepolyethylene glycol may be linear or branched. The polyethylene glycolmay have a molecular weight of about 20 kD, 30 kD, or 40 kD. Thepolyethylene glycol may be monomethoxy-PEG propionaldehyde. The patientupon which the polypeptide is administered may be a mammal, such as ahuman. The second polypeptide may be an Interferon molecule, such asInterferon-alpha, Interferon-beta, or Interferon-gamma, anothertherapeutic agent, such as IL-2 or IL-21, or combination thereof.

The present invention also provides a method of inhibiting at least oneof the conditions or symptoms of multiple myeloma comprisingadministering to a patient in need thereof a therapeutically effectiveamount of a polypeptide having at least 95% sequence identity with asequence selected from the group of SEQ ID NOs:2, 4, 6, 13, 15, 17, 19,21, 23, 25, 27, 29, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129,131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,159, 161, 163, 165, 167, 169, 171, 173 and 175, wherein the at least oneof the conditions or symptoms is selected from the group of back pain,loss of height, anaemia, kidney damage, repeated respiratory infections,and hypercalcaemia. The polypeptide may have at least 15, at least 30,at least 45, or at least 60 sequential amino acids to SEQ ID NOs:2, 4,6, 13, 15, 17, 19, 21, 23, 25, 27, 29, 41, 75, 77, 79, 81, 83, 85, 87,89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117,123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149,151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173 and 175. Thepolypeptide may further optionally include a polyethylene glycol moiety,which can be covalently linked to the polypeptide (e.g.,amino-terminally). The polyethylene glycol may be linear or branched.The polyethylene glycol may have a molecular weight of about 20 kD, 30kD, or 40 kD. The polyethylene glycol may be monomethoxy-PEGpropionaldehyde. The patient upon which the polypeptide is administeredmay be a mammal, such as a human.

The present invention also provides a method of inhibiting at least oneof the conditions or symptoms of multiple myeloma comprisingadministering to a patient in need thereof a therapeutically effectiveamount of a formulation comprising: a polypeptide having at least 90% or95% sequence identity with a sequence selected from the group of SEQ IDNOs:2, 4, 6, 13, 15, 17, 19, 21, 23, 25, 27, 29, 41, 75, 77, 79, 81, 83,85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115,117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147,149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173 and 175;a second polypeptide; and a pharmaceutically acceptable vehicle; whereinthe at least one of the conditions or symptoms is selected from thegroup of back pain, loss of height, anaemia, kidney damage, repeatedrespiratory infections, and hypercalcaemia. The polypeptide may have atleast 15, at least 30, at least 45, or at least 60 sequential aminoacids to SEQ ID NOs:2, 4, 6, 13, 15, 17, 19, 21, 23, 25, 27, 29, 41, 75,77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141,143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169,171, 173 and 175. The polypeptide may further optionally include apolyethylene glycol moiety, which can be covalently linked to thepolypeptide (e.g., amino-terminally). The polyethylene glycol may belinear or branched. The polyethylene glycol may have a molecular weightof about 20 kD, 30 kD, or 40 kD. The polyethylene glycol may bemonomethoxy-PEG propionaldehyde. The patient upon which the polypeptideis administered may be a mammal, such as a human. The second polypeptidemay be an Interferon molecule, such as Interferon-alpha,Interferon-beta, or Interferon-gamma, another therapeutic agent, such asIL-2 or IL-21, or combination thereof.

The present invention also provides a method of inhibiting at least oneof the conditions or symptoms of head and neck tumours comprisingadministering to a patient in need thereof a therapeutically effectiveamount of a polypeptide having at least 90% or 95% sequence identitywith a sequence selected from the group of SEQ ID NOs:2, 4, 6, 13, 15,17, 19, 21, 23, 25, 27, 29, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93,95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127,129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155,157, 159, 161, 163, 165, 167, 169, 171, 173 and 175, wherein the atleast one of the conditions or symptoms is selected from the group of anulcer or sore area in the head or neck that does not heal within a fewweeks, difficulty in swallowing, trouble with breathing or speaking, anumb feeling in the mouth, nose bleeds, persistent earache, difficultyin hearing, and swelling or lump in the mouth or neck. The polypeptidemay have at least 15, at least 30, at least 45, or at least 60sequential amino acids to SEQ ID NOs:2, 4, 6, 13, 15, 17, 19, 21, 23,25, 27, 29, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133,135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161,163, 165, 167, 169, 171, 173 and 175. The polypeptide may furtheroptionally include a polyethylene glycol moiety, which can be covalentlylinked to the polypeptide (e.g., amino-terminally). The polyethyleneglycol may be linear or branched. The polyethylene glycol may have amolecular weight of about 20 kD, 30 kD, or 40 kD. The polyethyleneglycol may be monomethoxy-PEG propionaldehyde. The patient upon whichthe polypeptide is administered may be a mammal, such as a human.

The present invention also provides a method of inhibiting at least oneof the conditions or symptoms of head and neck tumours comprisingadministering to a patient in need thereof a therapeutically effectiveamount of a formulation comprising: a polypeptide having at least 90% or95% sequence identity with a sequence selected from the group of SEQ IDNOs:2, 4, 6, 13, 15, 17, 19, 21, 23, 25, 27, 29, 41, 75, 77, 79, 81, 83,85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115,117, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147,149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173 and 175;a second polypeptide; and a pharmaceutically acceptable vehicle; whereinthe at least one of the conditions or symptoms is selected from thegroup of an ulcer or sore area in the head or neck that does not healwithin a few weeks, difficulty in swallowing, trouble with breathing orspeaking, a numb feeling in the mouth, nose bleeds, persistent earache,difficulty in hearing, and swelling or lump in the mouth or neck. Thepolypeptide may have at least 15, at least 30, at least 45, or at least60 sequential amino acids to SEQ ID NOs:2, 4, 6, 13, 15, 17, 19, 21, 23,25, 27, 29, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 109, 111, 113, 115, 117, 123, 125, 127, 129, 131, 133,135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161,163, 165, 167, 169, 171, 173 and 175. The polypeptide may furtheroptionally include a polyethylene glycol moiety, which can be covalentlylinked to the polypeptide (e.g., amino-terminally). The polyethyleneglycol may be linear or branched. The polyethylene glycol may have amolecular weight of about 20 kD, 30 kD, or 40 kD. The polyethyleneglycol may be monomethoxy-PEG propionaldehyde. The patient upon whichthe polypeptide is administered may be a mammal, such as a human. Thesecond polypeptide may be an Interferon molecule, such asInterferon-alpha, Interferon-beta, or Interferon-gamma, anothertherapeutic agent, such as IL-2 or IL-21, or combination thereof.

There are four main types of malignant melanoma which occur in the skin.These are known as cutaneous melanoma:

Superficial spreading melanoma is the most common type of melanoma.About 7 out of 10 (70%) are this type. They occur mostly in middle-agedpeople. The most common place in women is on the legs, while in men itis more common on the trunk, particularly the back. They tend to startby spreading out across the surface of the skin: this is known as theradial growth phase. If the melanoma is removed at this stage there is avery high chance of cure. If the melanoma is not removed, it will startto grow down deeper into the layers of the skin. There is then a riskthat it will spread in the bloodstream or lymph system to other parts ofthe body. Nodular melanoma occurs most often on the chest or back. It ismost commonly found in middle-aged people. It tends to grow deeper intothe skin quite quickly if it is not removed. This type of melanoma isoften raised above the rest of the skin surface and feels like a bump.It may be very dark brown-black or black. Lentigo maligna melanoma ismost commonly found on the face, particularly in older people. It growsslowly and may take several years to develop. Acral melanoma is usuallyfound on the palms of the hands, soles of the feet or around thetoenails. Other very rare types of melanoma of the skin includeamelanotic melanoma (in which the melanoma loses its pigment and appearsas a white area) and desmoplastic melanoma (which contains fibrous scartissue). Malignant melanoma can start in parts of the body other thanthe skin but this is very rare. The parts of the body that may beaffected are the eye, the mouth, under the fingernails (known assubungual melanoma) the vulval or vaginal tissues, or internally(cancerbacup internet website).

Most melanomas start with a change in the appearance of normal skin.This can look like an abnormal new mole. Less than a third develop inexisting moles. It can be difficult to tell the difference between amole and a melanoma, but the following checklist can be used to help. Itis known as the ABCD list. Asymmetry—Ordinary moles are usuallysymmetrical in shape. Melanomas are likely to be irregular orasymmetrical. Border—Moles usually have a well-defined regular border.Melanomas are more likely to have an irregular border with jagged edges.Colour—Moles are usually a uniform brown. Melanomas tend to have morethan one colour. They may be varying shades of brown mixed with black,red, pink, white or a bluish tint. Diameter—Moles are normally no biggerthan the blunt end of a pencil (about 6 mm across). Melanomas areusually more than 7 mm in diameter. Normal moles can be raised up fromthe skin and/or may be hairy. Itching, crusting or bleeding may alsooccur in melanomas—these are less common signs but should not be ignored(cancerbacup internet website). The effects of an IL-28 or IL-29polypeptide, fragment, or fusion protein on tumor response can beevaluated in a murine melanoma model similar to that described inHermans et al., Cancer Res. 2003 Dec. 1; 63(23):8408-13; Ramont et al.,Exp Cell Res. 2003 Nov. 15; 291(1):1-10; Safwat et al., J Exp TherOncol. 2003 July-August; 3(4):161-8; and Fidler, I. J., Nat New Biol.1973 Apr. 4; 242(118):148-9.

Chronic myeloid leukaemia (CML) is a rare type of cancer affectingmostly adults. It is a cancer of granulocytes (one of the main types ofwhite blood cells). In CML too many granulocytes are produced and theyare released into the blood when they are immature and unable to workproperly. The immature white blood cells are known as blasts. Theproduction of other types of blood cells is also disrupted. Normally,white blood cells repair and reproduce themselves in an orderly andcontrolled manner, but in chronic myeloid leukaemia the process gets outof control and the cells continue to divide and mature abnormally. Thedisease usually develops very slowly, which is why it is called‘chronic’ myeloid leukaemia (cancerbacup internet website).

Because CML develops (progresses) slowly, it is difficult to detect inits early stages. Sometimes it is discovered only when a blood test isdone for another reason. The symptoms of CML are often vague andnon-specific and are caused by the increased number of abnormal whiteblood cells in the bone marrow and the reduced number of normal bloodcells: a feeling of fullness or a tender lump on the left side of theabdomen. This is because, in CML, the spleen can become enlarged. Thespleen is an organ which lies just below the ribs on the left side ofthe abdomen. It filters the blood and removes worn-out red blood cells.The swelling of the spleen may also cause pressure on the stomach, whichcan lead to indigestion and poor appetite some people feel tired andlook pale, due to a lack of red blood cells (anaemia) due to a lowernumber of platelets in the blood some people may notice that they bleedor bruise more easily. As well as bruising more easily than normal, aspecial type of bruising can be seen. This consists of small blood-likespots usually seen on the legs or in the mouth and is called petechiae.Women may find that their periods become very much heavier. However,these symptoms and signs are rare some people may notice a generaliseditching. Chronic myeloid leukaemia can occur at any age, but it morecommonly affects middle-aged and older people. It is rare in children(cancerbacup internet website). The effects of an IL-28 or IL-29polypeptide, fragment, or fusion protein on tumor response can beevaluated in a murine chronic myeloid leukaemia model similar to thatdescribed in Ren, R., Oncogene. 2002 Dec. 9; 21(56):8629-42; Wertheim etal., Oncogene. 2002 Dec. 9; 21(56):8612-28; and Wolff et al., Blood.2001 Nov. 1; 98(9):2808-16.

Non-Hodgkin's lymphomas are a type of cancer of the lymphatic system.There are two main types of lymphoma. One is called Hodgkin's disease(named after Dr Hodgkin, who first described it). The other is callednon-Hodgkin's lymphoma. There are about 20 different types ofnon-Hodgkin's lymphoma. In most cases of Hodgkin's disease, a particularcell known as the Reed-Sternberg cell is found in the biopsies. Thiscell is not usually found in other lymphomas, so they are callednon-Hodgkin's lymphoma. This may not seem a very big difference, but itis important because the treatment for Hodgkin's and non-Hodgkin'slymphomas can be very different (cancerbacup internet website).

Often, the first sign of a non-Hodgkin's lymphoma is a painless swellingof a lymph node in the neck, armpit or groin. Other symptoms may includeany of the following: night sweats or unexplained high temperatures(fever); loss of appetite, unexplained weight loss and excessivetiredness; children may develop a cough or breathlessness. They may alsocomplain of abdominal pain or you may notice a lump in your child'sabdomen persistent itching of the skin all over the body (cancerbacupinternet website). The effects of an IL-28 or IL-29 polypeptide,fragment, or fusion protein on tumor response can be evaluated in amurine non-Hodgkin's lymphoma model similar to that described in Ansellet al., Leukemia. 2004 March; 18(3):616-23; De Jonge et al., J Immunol.1998 Aug. 1; 161(3):1454-61; and Slavin et al., Nature. 1978 Apr. 13;272(5654):624-6.

Renal cell carcinoma, a form of kidney cancer that involves cancerouschanges in the cells of the renal tubule, is the most common type ofkidney cancer in adults. Why the cells become cancerous is not known. Ahistory of smoking greatly increases the risk for developing renal cellcarcinoma. Some people may also have inherited an increased risk todevelop renal cell carcinoma, and a family history of kidney cancerincreases the risk. People with von Hippel-Lindau disease, a hereditarydisease that affects the capillaries of the brain, commonly also developrenal cell carcinoma. Kidney disorders that require dialysis fortreatment also increase the risk for developing renal cell carcinoma.The first symptom is usually blood in the urine. Sometimes both kidneysare involved. The cancer metastasizes or spreads easily, most often tothe lungs and other organs, and about one-third of patients havemetastasis at the time of diagnosis (Medline Plus Medical EncyclopediaInternet website). The effects of an IL-28 or IL-29 polypeptide,fragment, or fusion protein on tumor response can be evaluated in amurine renal cell carcinoma model similar to that described in Sayers etal., Cancer Res. 1990 Sep. 1; 50(17):5414-20; Salup et al., Immunol.1987 Jan. 15; 138(2):641-7; and Luan et al., Transplantation. 2002 May27; 73(10):1565-72.

The cervix is the neck of the uterus that opens into the vagina.Cervical cancer, also called cervical carcinoma, develops from abnormalcells on the surface of the cervix. Cervical cancer is one of the mostcommon cancers affecting women. Cervical cancer is usually preceded bydysplasia, precancerous changes in the cells on the surface of thecervix. These abnormal cells can progress to invasive cancer. Once thecancer appears it can progress through four stages. The stages aredefined by the extent of spread of the cancer. The more the cancer hasspread, the more extensive the treatment is likely to be. There are 2main types of cervical cancer: (1) Squamous type (epidermoid cancer):This is the most common type, accounting for about 80% to 85% ofcervical cancers. This cancer may be caused by sexually transmitteddiseases. One such sexual disease is the human papillomavirus, whichcauses venereal warts. The cancerous tumor grows on and into the cervix.This cancer generally starts on the surface of the cervix and may bediagnosed at an early stage by a Pap smear. (2) Adenocarcinoma: Thistype of cervical cancer develops from the tissue in the cervical glandsin the canal of the cervix. Early cervical cancer usually causes nosymptoms. The cancer is usually detected by a Pap smear and pelvic exam.This is why you should start having Pap smears and pelvic exams as soonas you become sexually active. Healthy young women who have never beensexually active should have their first annual pelvic exam by age 18.Later stages of cervical cancer cause abnormal vaginal bleeding or abloodstained discharge at unexpected times, such as between menstrualperiods, after intercourse, or after menopause. Abnormal vaginaldischarge may be cloudy or bloody or may contain mucus with a bad odor.Advanced stages of the cancer may cause pain (University of MichiganHealth System Internet website). The effects of an IL-28 or IL-29polypeptide, fragment, or fusion protein on tumor response can beevaluated in a murine cervical cancer model similar to that described inAhn et al., Hum Gene Ther. 2003 Oct. 10; 14(15):1389-99; Hussain et al.,Oncology. 1992; 49(3):237-40; and Sengupta et al., Oncology. 1991;48(3):258-61.

Most cancers of the head and neck are of a type called carcinoma (inparticular squamous cell carcinoma). Carcinomas of the head and neckstart in the cells that form the lining of the mouth, nose, throat orear, or the surface layer covering the tongue. However, cancers of thehead and neck can develop from other types of cells. Lymphoma developsfrom the cells of the lymphatic system. Sarcoma develops from thesupportive cells which make up muscles, cartilage or blood vessels.Melanoma starts from cells called melanocytes, which give colour to theeyes and skin. The symptoms of a head and neck cancer will depend onwhere it is—for example, cancer of the tongue may cause some slurring ofspeech. The most common symptoms are an ulcer or sore area in the heador neck that does not heal within a few weeks; difficulty in swallowing,or pain when chewing or swallowing; trouble with breathing or speaking,such as persistent noisy breathing, slurred speech or a hoarse voice; anumb feeling in the mouth; a persistent blocked nose, or nose bleeds;persistent earache, ringing in the ear, or difficulty in hearing; aswelling or lump in the mouth or neck; pain in the face or upper jaw; inpeople who smoke or chew tobacco, pre-cancerous changes can occur in thelining of the mouth, or on the tongue. These can appear as persistentwhite patches (leukoplakia) or red patches (erythroplakia). They areusually painless but can sometimes be sore and may bleed (CancerbacupInternet website). The effects of an IL-28 or IL-29 polypeptide,fragment, or fusion protein on tumor response can be evaluated in amurine head and neck tumor model similar to that described in Kuriakoseet al., Head Neck. 2000 January; 22(1):57-63; Cao et al., Clin CancerRes. 1999 July; 5(7):1925-34; Hier et al., Laryngoscope. 1995 October;105(10):1077-80; Braakhuis et al., Cancer Res. 1991 Jan. 1; 51(1):211-4;Baker, S. R., Laryngoscope. 1985 January; 95(1):43-56; and Dong et al.,Cancer Gene Ther. 2003 February; 10(2):96-104.

Papillary and follicular thyroid cancers account for 80 to 90 percent ofall thyroid cancers. Both types begin in the follicular cells of thethyroid. Most papillary and follicular thyroid cancers tend to growslowly. If they are detected early, most can be treated successfully.Medullary thyroid cancer accounts for 5 to 10 percent of thyroid cancercases. It arises in C cells, not follicular cells. Medullary thyroidcancer is easier to control if it is found and treated before it spreadsto other parts of the body. Anaplastic thyroid cancer is the leastcommon type of thyroid cancer (only 1 to 2 percent of cases). It arisesin the follicular cells. The cancer cells are highly abnormal anddifficult to recognize. This type of cancer is usually very hard tocontrol because the cancer cells tend to grow and spread very quickly.Early thyroid cancer often does not cause symptoms. But as the cancergrows, symptoms may include: A lump, or nodule, in the front of the necknear the Adam's apple; Hoarseness or difficulty speaking in a normalvoice; Swollen lymph nodes, especially in the neck; Difficultyswallowing or breathing; or Pain in the throat or neck (National CancerInstitute's Internet website). The effects of an IL-28 or IL-29polypeptide, fragment, or fusion protein on tumor response can beevaluated in a murine or rat thyroid tumor model similar to thatdescribed in Quidville et al., Endocrinology. 2004 May; 145(5):2561-71(mouse model); Cranston et al., Cancer Res. 2003 Aug. 15; 63(16):4777-80(mouse model); Zhang et al., Clin Endocrinol (Oxf). 2000 June;52(6):687-94 (rat model); and Zhang et al., Endocrinology. 1999 May;140(5):2152-8 (rat model).

Tumors that begin in brain tissue are known as primary tumors of thebrain. Primary brain tumors are named according to the type of cells orthe part of the brain in which they begin. The most common primary braintumors are gliomas. They begin in glial cells. There are many types ofgliomas. (1) Astrocytoma—The tumor arises from star-shaped glial cellscalled astrocytes. In adults, astrocytomas most often arise in thecerebrum. In children, they occur in the brain stem, the cerebrum, andthe cerebellum. A grade III astrocytoma is sometimes called ananaplastic astrocytoma. A grade IV astrocytoma is usually called aglioblastoma multiforme. (2) Brain stem glioma—The tumor occurs in thelowest part of the brain. Brain stem gliomas most often are diagnosed inyoung children and middle-aged adults. (3) Ependymoma—The tumor arisesfrom cells that line the ventricles or the central canal of the spinalcord. They are most commonly found in children and young adults. (4)Oligodendroglioma—This rare tumor arises from cells that make the fattysubstance that covers and protects nerves. These tumors usually occur inthe cerebrum. They grow slowly and usually do not spread intosurrounding brain tissue. They are most common in middle-aged adults.The symptoms of brain tumors depend on tumor size, type, and location.Symptoms may be caused when a tumor presses on a nerve or damages acertain area of the brain. They also may be caused when the brain swellsor fluid builds up within the skull. These are the most common symptomsof brain tumors: Headaches (usually worse in the morning); Nausea orvomiting; Changes in speech, vision, or hearing; Problems balancing orwalking; Changes in mood, personality, or ability to concentrate;Problems with memory; Muscle jerking or twitching (seizures orconvulsions); and Numbness or tingling in the arms or legs (NationalCancer Institute's Internet website). The effects of an IL-28 or IL-29polypeptide, fragment, or fusion protein on tumor response can beevaluated in a glioma animal model similar to that described inSchueneman et al., Cancer Res. 2003 Jul. 15; 63(14):4009-16; Martinet etal., Eur J Surg Oncol. 2003 May; 29(4):351-7; Bello et al., Clin CancerRes. 2002 November; 8(11):3539-48; Ishikawa et al., Cancer Sci. 2004January; 95(1):98-103; Degen et al., J Neurosurg. 2003 November;99(5):893-8; Engelhard et al., Neurosurgery. 2001 March; 48(3):616-24;Watanabe et al., Neurol Res. 2002 July; 24(5):485-90; and Lumniczky etal., Cancer Gene Ther. 2002 January; 9(1):44-52.

Multiple myeloma is a type of cancer. It affects certain white bloodcells called plasma cells. When cancer involves plasma cells, the bodykeeps producing more and more of these cells. The unneeded plasmacells—all abnormal and all exactly alike—are called myeloma cells.Myeloma cells tend to collect in the bone marrow and in the hard, outerpart of bones. Sometimes they collect in only one bone and form a singlemass, or tumor, called a plasmacytoma. In most cases, however, themyeloma cells collect in many bones, often forming many tumors andcausing other problems. When this happens, the disease is calledmultiple myeloma.

Myeloma cells tend to collect in the bone marrow and in the hard, outerpart of bones. Sometimes they collect in only one bone and form a singlemass, or tumor, called a plasmacytoma. In most cases, however, themyeloma cells collect in many bones, often forming many tumors andcausing other problems. When this happens, the disease is calledmultiple myeloma. Because people with multiple myeloma have anabnormally large number of identical plasma cells, they also have toomuch of one type of antibody. These myeloma cells and antibodies cancause a number of serious medical problems: (1) As myeloma cellsincrease in number, they damage and weaken bones, causing pain andsometimes fractures. Bone pain can make it difficult for patients tomove; (2) When bones are damaged, calcium is released into the blood.This may lead to hypercalcemia—too much calcium in the blood.Hypercalcemia can cause loss of appetite, nausea, thirst, fatigue,muscle weakness, restlessness, and confusion; (3) Myeloma cells preventthe bone marrow from forming normal plasma cells and other white bloodcells that are important to the immune system. Patients may not be ableto fight infection and disease; (4) The cancer cells also may preventthe growth of new red blood cells, causing anemia. Patients with anemiamay feel unusually tired or weak; and (5) Multiple myeloma patients mayhave serious problems with their kidneys. Excess antibody proteins andcalcium can prevent the kidneys from filtering and cleaning the bloodproperly. Symptoms of multiple myeloma depend on how advanced thedisease is. In the earliest stage of the disease, there may be nosymptoms. When symptoms do occur, patients commonly have bone pain,often in the back or ribs. Patients also may have broken bones,weakness, fatigue, weight loss, or repeated infections. When the diseaseis advanced, symptoms may include nausea, vomiting, constipation,problems with urination, and weakness or numbness in the legs (NationalCancer Institute's Internet website). The effects of an IL-28 or IL-29polypeptide, fragment, or fusion protein on tumor response can beevaluated in a multiple myeloma murine model similar to that describedin Oyajobi et al., Blood. 2003 Jul. 1; 102(1):311-9; Croucher et al., JBone Miner Res. 2003 March; 18(3):482-92; Asosingh et al., Hematol J.2000; 1(5):351-6; and Miyakawa et al., Biochem Biophys Res Commun. 2004Jan. 9; 313(2):258-62.

The effects of an IL-28 or IL-29 polypeptide, fragment, or fusionprotein on tumor response can be evaluated in a human small/non-smallcell lung carcinoma xenograft model. Briefly, human tumors are graftedinto immunodeficient mice and these mice are treated with IL-28 or IL-29polypeptide, fragment, or fusion proteins alone or in combination withother agents which can be used to demonstrate the efficacy of thetreatment by evaluating tumor growth (Nemati et al., Clin Cancer Res.2000 May; 6(5):2075-86; and Hu et al., Clin Cancer Res. 2004 Nov. 15;10(22):7662-70).

There are two different types of primary liver cancer. The most commonkind is called hepatoma or hepatocellular carcinoma (HCC), and arisesfrom the main cells of the liver (the hepatocytes). This type is usuallyconfined to the liver, although occasionally it spreads to other organs.It occurs mostly in people with a liver disease called cirrhosis. Thereis also a rarer sub-type of hepatoma called Fibrolamellar hepatoma,which may occur in younger people and is not related to previous liverdisease. The other type of primary liver cancer is calledcholangiocarcinoma or bile duct cancer, because it starts in the cellslining the bile ducts. Most people who develop hepatoma usually alsohave a condition called cirrhosis of the liver. This is a fine scarringthroughout the liver which is due to a variety of causes includinginfection and heavy alcohol drinking over a long period of time.However, only a small proportion of people who have cirrhosis of theliver develop primary liver cancer. Infection with either the hepatitisB or hepatitis C virus can lead to liver cancer, and can also be thecause of cirrhosis, which increases the risk of developing hepatoma.People who have a rare condition called haemochromatosis, which causesexcess deposits of iron in the body, have a higher chance of developinghepatoma. Thus, the IL-28 and IL-29 polypeptides, fragments, and/orfusion proteins of the present invention may be used to treat, prevent,inhibit the progression of, delay the onset of, and/or reduce theseverity or inhibit at least one of the conditions or symptomsassociated with hepatocellular carcinoma. The hepatocellular carcinomamay or may not be associated with an hepatitis (e.g., hepatitis A,hepatitis B, hepatitis C and hepatitis D) infection.

The effects of an IL-28 or IL-29 polypeptide, fragment, or fusionprotein on tumor response can be evaluated in a hepatocellular carcinomatransgenic mouse model, which includes the overexpression oftransforming growth factor-α (TFG-α) alone (Jhappan et al., Cell,61:1137-1146 (1990); Sandgren et al., Mol. Cell Biol., 13:320-330(1993); Sandgren et al., Oncogene, 4:715-724 (1989); and Lee et al.,Cancer Res., 52:5162:5170 (1992)) or in combination with c-myc (Murakamiet al., Cancer Res., 53:1719-1723 (1993), mutated H-ras (Saitoh et al.,Oncogene, 5:1195-2000 (1990)), hepatitis B viral genes encoding HbsAgand HBx (Toshkov et al., Hepatology, 20:1162-1172 (1994) and Koike etal., Hepatology, 19:810-819 (1994)), SV40 large T antigen (Sepulveda etal., Cancer Res., 49:6108-6117 (1989) and Schirmacher et al., Am. J.Pathol., 139:231-241 (1991)) and FGF19 (Nicholes et al., AmericanJournal of Pathology, 160(6):2295-2307 (June 2002)).

The IL-28 and IL-29 polypeptides, proteins, fusions and fragmentsthereof of the present invention can be used in combination with otheragents in treating, preventing, inhibiting the progression of, delayingthe onset of, and/or reducing at least one of the conditions or symptomsassociated with hepatocellular carcinoma, such other agents includechemotherapeutic agents (e.g., adriamycin, 5-FU), Bevacizumab,erlotinib, Lapatinib, Doxorubicin, bortezomib, Thalidomide, Gemcitabine,Oxaliplatin, and epirubicin.

The powerful inducer of apoptosis Apo2L/TNF-related apoptosis-inducingligand (TRAIL) has generated exciting promise as a potential tumourspecific cancer therapeutic agent, since it selectively inducesapoptosis in transformed versus normal cells. Interferons (IFNs) areimportant modulators of TRAIL expression, thus the ligand appears toplay an important role in surveillance against viral infection andmalignant transformation. Fiorucci et al., Curr Pharm Des. 2005;11(7):933-44. IL-28 and IL-29 also appear to be important regulators ofTRAIL (See Example 41 where TRAIL is upregulated by IL-29).

The IL-28 and IL-29 polypeptides, proteins, fusions and fragmentsthereof of the present invention can be used in combination with otheragents in treating, preventing, inhibiting the progression of, delayingthe onset of, and/or reducing at least one of the conditions or symptomsassociated with prostate cancer such as radiation therapy, chemotherapy(e.g., docetaxel, pacletaxel, estramustine, mitoxantrone), estramustine,docetaxel, Paclitaxel. Estramustine/etoposide. Estramustine/vinblastine,Estramustine/paclitaxel, mitoxantrone, zoledronate, prednisolone,celecoxib, etoposide, Erlotiib, Lapatinib and Sorafenib.

The IL-28 and IL-29 polypeptides, proteins, fusions and fragmentsthereof of the present invention can be used in combination with otheragents in treating, preventing, inhibiting the progression of, delayingthe onset of, and/or reducing at least one of the conditions or symptomsassociated with pancreatic carcinoma such as fluorouracil (5-FU)chemotherapy, radiation therapy, gemcitabine, fluorouracil, oxaliplatin,irinotecan, leucovorin, capecitabine, Dalteparin, Bevacizumab, andTarceva.

The IL-28 and IL-29 polypeptides, proteins, fusions and fragmentsthereof of the present invention can be used in combination with otheragents in treating, preventing, inhibiting the progression of, delayingthe onset of, and/or reducing at least one of the conditions or symptomsassociated with colorectal carcinoma such as folic acid, fluorouracil[5-FU], irinotecan, leucovorin, irinotecan, levamisole, bevacizumab,IFL-bevacizumab, and cetuximab.

The IL-28 and IL-29 polypeptides, proteins, fusions and fragmentsthereof of the present invention can be used in combination with otheragents in treating, preventing, inhibiting the progression of, delayingthe onset of, and/or reducing at least one of the conditions or symptomsassociated with renal cell carcinoma such as kinase inhibitors (e.g.,sutininib and sorafenib), IL-2, interferon-alpha, and Vinblastine.

The IL-28 and IL-29 polypeptides, proteins, fusions and fragmentsthereof of the present invention can be used in combination with otheragents in treating, preventing, inhibiting the progression of, delayingthe onset of, and/or reducing at least one of the conditions or symptomsassociated with melanoma such as Interferon alpha 2b, dacarbazine(DTIC), nitrosoureas, carmustine (BCNU), lomustine, tamoxifen,cisplatin, and IL-2.

The IL-28 and IL-29 polypeptides, proteins, fusions and fragmentsthereof of the present invention can be used in combination with otheragents in treating, preventing, inhibiting the progression of, delayingthe onset of, and/or reducing at least one of the conditions or symptomsassociated with esophageal cancer such as chemotherapeutica agents(e.g., Cisplatin, bleomycin, SFU, paclitaxel, VP-16; irinotecan,Interferon-alpha, taxotere, gemcitabine, venorelbine, carboplatin,mitomycin-C, doxorubicin (adriamycin), methotrexate, Methyl-GAG).

The IL-28 and IL-29 polypeptides, proteins, fusions and fragmentsthereof of the present invention can be used in combination with otheragents in treating, preventing, inhibiting the progression of, delayingthe onset of, and/or reducing at least one of the conditions or symptomsassociated with ovarian epithelial carcinoma such as chemotherapeuticaagents (paclitaxel, cisplatin, carboplatin, paclitaxel, doxorubicin,cyclophosphamide), Topotecan, Liposomal doxorubicin and topotecan,Gemcitabine, Fluorouracil and leucovorin, Tamoxifen, Etoposide,Ifosfamide, Hexamethylmelamine (HMM), and Capecitabine.

The IL-28 and IL-29 polypeptides, proteins, fusions and fragmentsthereof of the present invention can be used in combination with otheragents in treating, preventing, inhibiting the progression of, delayingthe onset of, and/or reducing at least one of the conditions or symptomsassociated with Non-small cell lung carcinoma such as cisplatin,carboplatin, mitomycin, paclitaxel, docetaxel, topotecan, irinotecan,vinorelebine, vinorelbine, gemcitabine, IRESSA, TARCEVA, bevacizumab,Sorafenib, Celecoxib,

The Use of IL-28 and IL-29 to Treat Autoimmune Disorders

The present invention provides for a method of treating, preventing,inhibiting the progression of, delaying the onset of, and/or reducing atleast one of the conditions or symptoms associated with autoimmunedisorder comprising administering to a patient in need thereof atherapeutically effective amount of a polypeptide selected from thegroup of SEQ ID NOs:2, 4, 6, 13, 15, 17, 19, 21, 23, 25, 27, 29, 36, 37,38, 39, 40, 41, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129,131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,and 161 wherein the autoimmune disorder is selected from the group ofselected from the group of multiple sclerosis, arthritis, rheumatoidarthritis, inflammatory bowel disease, systemic lupus erythematosus, andpsoriasis. The polypeptide may further optionally include a polyethyleneglycol moiety, which can be covalently linked to the polypeptide (e.g.,amino-terminally). The polyethylene glycol may be linear or branched.The polyethylene glycol may have a molecular weight of about 20 kD, 30kD, or 40 kD. The polyethylene glycol may be monomethoxy-PEGpropionaldehyde. The patient upon which the polypeptide is administeredmay be a mammal, such as a human.

The present invention provides for a method of treating an autoimmunedisorder comprising administering to a patient in need thereof atherapeutically effective amount of a polypeptide selected from thegroup of SEQ ID NOs:163, 165, 167, 169, 171, 173 and 175, wherein theautoimmune disorder is selected from the group of multiple sclerosis,arthritis, rheumatoid arthritis, ulcerative colitis, Crohn's disease,systemic lupus erythematosus, and psoriasis.

The present invention provides for a method of treating an autoimmunedisorder comprising administering to a patient in need thereof atherapeutically effective amount of a polypeptide having at least 95%sequence identity with a sequence selected from the group of SEQ IDNOs:163, 165, 167, 169, 171, 173 and 175, wherein the autoimmunedisorder is selected from the group of multiple sclerosis, arthritis,rheumatoid arthritis, ulcerative colitis, Crohn's disease, systemiclupus erythematosus, and psoriasis.

The present invention provides for a a method of an autoimmune disordercomprising administering to a patient in need thereof a therapeuticallyeffective amount of a formulation comprising: a polypeptide having atleast 95% sequence identity with a sequence selected from the group ofSEQ ID NOs:163, 165, 167, 169, 171, 173 and 175; a second polypeptide; apharmaceutically acceptable vehicle; and wherein the cancer is selectedfrom the group of multiple sclerosis, arthritis, rheumatoid arthritis,ulcerative colitis, Crohn's disease, systemic lupus erythematosus, andpsoriasis.

The present invention provides for a method of inhibiting theprogression of an autoimmune disorder comprising administering to apatient in need thereof a therapeutically effective amount of aformulation comprising: a polypeptide having at least 95% sequenceidentity with a sequence selected from the group of SEQ ID NOs:163, 165,167, 169, 171, 173 and 175; a second polypeptide; a pharmaceuticallyacceptable vehicle; and wherein the cancer is selected from the group ofmultiple sclerosis, arthritis, rheumatoid arthritis, ulcerative colitis,Crohn's disease, systemic lupus erythematosus, and psoriasis.

The present invention provides for a method of delaying a multiplesclerosis relapse in a patient comprising administering to the patientin need thereof a therapeutically effective amount of a formulationcomprising: a polypeptide having at least 95% sequence identity with asequence selected from the group of SEQ ID NOs:163, 165, 167, 169, 171,173 and 175; a pharmaceutically acceptable vehicle.

The present invention provides for a method of reducing the severity ofa multiple sclerosis relapse in a patient comprising administering tothe patient in need thereof a therapeutically effective amount of aformulation comprising: a polypeptide having at least 95% sequenceidentity with a sequence selected from the group of SEQ ID NOs:163, 165,167, 169, 171, 173 and 175; a pharmaceutically acceptable vehicle.

Rheumatoid Arthritis

Rheumatoid arthritis is an autoimmune disorder where the immuneresponses of the body are targeted against the body's own proteins, inparticular collagen, a protein that is the foundation of multipletissues, specifically joints. The resulting immune response againstcollagen leads to destruction of the joints. Over time, the patient canlose the ability to move their fingers and toes and can experience acutepain in the joints and knees. Serum from arthritis patients haveincreased amounts of TNFα (tumor necrosis factor) and antibodies againstcollagen, both of which are not only indicators of chronic disease butalso contribute towards the pathology of the disease. (Smolen andStein+er G, Nat. Rev. Drug Discov., 2:473-488, 2003; Firestein, Nature423:356-361, 2003.) Furthermore, the disease is initiated and mediatedby CD4⁺ T cells. DCs present collagen as an antigen to CD4⁺ T cells. Thecollagen-induced arthritis (CIA) model is a mouse model for rheumatoidarthritis that reflects to large extent the disease seen in humans.(Moore, Methods Mol. Biol. 225:175-179, 2003: Waksman, Scand. J.Immunol., 56:12-34, 2002). Mice are immunized with 2 doses of collagenemulsified in CFA at the base of the tail. This results in swelling ofthe paws that increases over a period of time and can be both visuallyscored and measured using calipers. IL-28A, IL-28B, or IL-29 isadministered to groups of collagen-immunized mice, and effects ondisease scores are evaluated. Inhibition of paw scores and thickness byIL-28A, IL-28B, and IL-29 is indicative of it's inhibitory effect on anongoing autoimmune response.

Inflammatory Bowel Disease

Inflammation in the gut resulting from defective immune regulation,known as inflammatory bowel disease (IBD) is characterized into twobroad disease definitions, Crohn's disease (CD) and Ulcerative colitis(UC). Generally, CD is thought to be due to dysfunction in theregulation of Th1 responses, and UC is believed to be due to dysfunctionin the regulation of Th2 responses. Multiple cytokines, chemokines, andmatrix metaloproteinases have beens shown to be upregulated in inflamedlesions from IBD patients. These include IL-1, IL-12, IL-18, IL-15,TNF-α, IFN-γ, MIP1α, MIP1β, and MIP2. Currently REMICADE® (Centocor,Malvern, Pa.) is the only drug that has successfully been used to targetthe disease itself when treating CD patients, with other treatmentsgenerally improving the quality of life of patients. IL-28A, IL-28B, andIL-29 inhibition of the autoimmune response associated with IBD isdemonstrated in IBD models, such as the mouse DSS, TNBS, CD4+CD45Rbhi,mdrla−/− and graft v. host disease (GVHD) intestinal inflammationmodels. (Stadnicki A and Colman R W, Arch Immunol Ther Exp 51:149-155,2003; Pizarro T T et al., Trends in Mol Med 9:218-222, 2003). Oneexperimental model for human IBD is the oral administration of dextransodium sulfate (DSS) to rodents. DSS induces both acute and chroniculcerative colitis with features somewhat resembling histologicalfindings in humans. Colitis induced by DSS involves gut bacteria,macrophages and neutrophils, with a minor role for T and B cells (Mahleret al., Am. J. Physiol. 274:G544-G551, 1998; Egger et al., Digestion62:240-248, 2000). TNBS-induced colitis is considered a Th1 mediateddisease and therefore resembles CD more than UC in humans. Tri-nitrobenzene sulfonic acid (TNBS) is infused into mice intra-rectally invarying doses (strain dependent) to induce antigen specific (TNBS) Tcell response that involves secretion of Th1-like cytokines IL-12, IL-18and IFNγ. Colitis involves recruitment of antigen-specific T cells,macrophages and neutrophils to the site of inflammation (Neurath et al.,Int. Rev. Immunol., 19:51-62, 2000; Dohi T et al., J. Exp. Med.189:1169-1179, 1999). Another relatively new model for colitis is theCD4+CD45RB^(hi) transfer model into SCID mice. CD4⁺ T cells can bedivided broadly into 2 categories based on expression of CD45Rb.CD4+CD45RB^(hi) cells are considered naïve T cells whereasCD4+CD45Rb^(lo) cells are considered regulatory T cells. Transfer ofwhole CD4⁺ T cells into syngenic SCID mice does not induce symptoms ofcolitis. However, if only the CD4+CD45RB^(hi) T cells are injected intoSCID mice, mice develop colitis over a period of 3-6 weeks. Co-transferof the CD4+CD45Rb^(lo) regulatory T cells along with the naïve T cellsinhibits colitis suggesting that the regulatory T cells play animportant role in regulating the immune response (Leach et al., Am. J.Pathol., 148:1503-1515, 1996; Powrie et al., J. Exp. Med., 179:589-600,1999). This model will demonstrate that IL-28A, IL-28B, and IL-29inhibit colitis by upregulating T regulatory function via its ability togenerate tolerogenic DCs in mice. A clinically relevant model of colitisassociated with bone marrow transplantation is GVHD-induced colitis.Graft-versus-host disease (GVHD) develops in immunoincompetent,histocompatible recipients of effector cells, which proliferate andattack host cells. Patients receiving allogeneic bone marrowtransplantation or severe aplastic anemia are at risk for GVHD. In bothmice and humans, diarrhea is a common and serious symptom of thesyndrome. In human, both colonic and small intestinal disease have beenobserved. Mouse models for GVHD-induced colitis show similarhistological disease as seen in humans. These mouse models can thereforebe used to assess the efficacy of colitis inhibiting drugs for GVHD(Eigenbrodt et al., Am. J. Pathol., 137:1065-1076, 1990; Thiele et al.,J. Clin. Invest., 84:1947-1956, 1989).

Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is an immune-complex related disordercharacterized by chronic IgG antibody production directed at ubiquitousself antigens (anti-dsDNA). The effects of SLE are systemic, rather thanlocalized to a specific organ. Multiple chromosomal loci have beenassociated with the disease and may contribute towards different aspectsof the disease, such as anti-dsDNA antibodies and glomerulonephritis.CD4⁺ T cells have been shown to play an active part in mouse models ofSLE (Horwitz, Lupus 10:319-320, 2001; Yellin and Thienel, Curr.Rheumatol. Rep., 2:24-37, 2000). The role for CD8⁺ T cells is notclearly defined, but there is evidence to suggest that “suppressor” CD8⁺T cell function is impaired in lupus patients (Filaci et al., J.Immunol., 166:6452-6457, 2001; Sakane et al, J. Immunol., 137:3809-3813,1986).

Sera from human SLE patients and mouse models are assayed for IL-28A,IL-28B, and IL-29 activity. CD8⁺ T cell suppressor activity in PBLs fromhuman SLE patients after culture with of IL-28A, IL-28B, or IL-29 isevaluated in vitro. Suppressor activity of CD8⁺ T cells from SLEpatients is evaluated by their ability to inhibit anti-CD3 inducedproliferation of autologous PBMC. Inhibition function correlates withsecretion of IFNγ and IL-6 in the cultures. Increased IFNγ and IL-6 incultures from IL-28A, IL-28B, or IL-29 treated patients might indicatehigher suppressor activity (Filaci et al., J. Immunol. 166:6452-6457,2001)

Psoriasis

Psoriasis is a chronic inflammatory skin disease that is associated withhyperplastic epidermal keratinocytes and infiltrating mononuclear cells,including CD4+ memory T cells, neutrophils and macrophages(Christophers, Int. Arch. Allergy Immunol., 110:199, 1996). It iscurrently believed that environmental antigens play a significant rolein initiating and contributing to the pathology of the disease. However,it is the loss of tolerance to self antigens that is thought to mediatethe pathology of psoriasis. Dendritic cells and CD4⁺ T cells are thoughtto play an important role in antigen presentation and recognition thatmediate the immune response leading to the pathology. A model ofpsoriasis based on the CD4+CD45RB transfer model was recently developed(Davenport et al., Internat. Immunopharmacol., 2:653-672 (2002)).IL-28A, IL-28B, or IL-29 is administered to mice that are injected withpsoriasis inducing cells and the effects on clinical score (skindisease) is evaluated, showing beneficial effects of IL-28A, IL-28B, andIL-29.

IL-28A, IL-28B, or IL-29 can be administered in combination with otheragents already in use in autoimmunity and/or cancer including agentssuch as interferon-alpha (IFN-α, e.g., PEGASYS®, PEG-INTRON®, INFERGEN®,Albuferon-Alpha™), interferon-beta (INF-β, e.g., AVONEX®, BETASERON®,REBIF®), interferon-gamma (IFNγ, e.g., ACTIMMUNE®), NOVANTRONE®,ENBREL®, REMICADE®, LEUKINE®, APO2L/TNF-Related Apoptosis-InducingLigand (TRAIL), IL-21 and IL-2. Establishing the optimal dose level andscheduling for IL-28A, IL-28B, and IL-29 is done by a variety of means,including study of the pharmacokinetics and pharmacodynamics of IL-28A,IL-28B, and IL-29; determination of effective doses in animal models,and evaluation of the toxicity of IL-28A, IL-28B, and IL-29. Directpharmacokinetic measurements done in primates and clinical trials canthen be used to predict theoretical doses in patients that achieveplasma IL-28A, IL-28B, and IL-29 levels that are of sufficient magnitudeand duration to achieve a biological response in patients.

The invention is further illustrated by the following non-limitingexample.

EXAMPLES Example 1 Mammalian Expression Plasmids

An expression plasmid containing zcyto20 and zcyto21 was constructed viahomologous recombination. Fragments of zcyto20 and zcyto21 cDNA weregenerated using PCR amplification. The primers for PCR were as follows:

zcyto20/pZMP21: zc40923, and zc43152 SEQ ID NOS: 42 and 43,respectively; and zcyto21/pZMP21: zc40922, and zc43153 SEQ ID NOS:72 and73, respectively.

The PCR reaction mixture was run on a 1% agarose gel and a bandcorresponding to the size of the insert was gel-extracted using aQIAquick™ Gel Extraction Kit (Qiagen, Valencia, Calif.).

The plasmid pZMP21, which was cut with BglII, was used for recombinationwith the PCR insert fragment. Plasmid pZMP21 is a mammalian expressionvector containing an expression cassette having the MPSV promoter, andmultiple restriction sites for insertion of coding sequences; an E. coliorigin of replication; a mammalian selectable marker expression unitcomprising an SV40 promoter, enhancer and origin of replication, a DHFRgene, and the SV40 terminator; and URA3 and CEN-ARS sequences requiredfor selection and replication in S. cerevisiae. It was constructed frompZP9 (deposited at the American Type Culture Collection, 10801University Boulevard, Manassas, Va. 20110-2209, under Accession No.98668) with the yeast genetic elements taken from pRS316 (deposited atthe American Type Culture Collection, 10801 University Boulevard,Manassas, Va. 20110-2209, under Accession No. 77145), an internalribosome entry site (IRES) element from poliovirus, and theextracellular domain of CD8 truncated at the C-terminal end of thetransmembrane domain.

One hundred microliters of competent yeast (S. cerevisiae) cells wereindependently combined with 10 μl of the insert DNA and 100 ng of thecut pZMP21 vector above, and the mix was transferred to a 0.2-cmelectroporation cuvette. The yeast/DNA mixture was electropulsed usingpower supply (BioRad Laboratories, Hercules, Calif.) settings of 0.75 kV(5 kV/cm), Go ohms, and 25 μF. Six hundred μl of 1.2 M sorbitol wasadded to the cuvette, and the yeast was plated in a 100-μl and 300 μlaliquot onto two URA-D plates and incubated at 30° C. After about 72hours, the Ura⁺ yeast transformants from a single plate were resuspendedin 1 ml H₂O and spun briefly to pellet the yeast cells. The cell pelletwas resuspended in 0.5 ml of lysis buffer (2% Triton X-100, 1% SDS, 100mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). The five hundred microliters ofthe lysis mixture was added to an Eppendorf tube containing 250 μlacid-washed glass beads and 300 μl phenol-chloroform, was vortexed for 3minutes, and spun for 5 minutes in an Eppendorf centrifuge at maximumspeed. Three hundred microliters of the aqueous phase was transferred toa fresh tube, and the DNA was precipitated with 600 μl ethanol (EtOH)and 30 μl 3M sodium acetate, followed by centrifugation for 30 minutesat maximum speed. The DNA pellet was resuspended in 30 μl TE.

Transformation of electrocompetent E. coli host cells (MC1061) was doneusing 5 μl of the yeast DNA prep and 50 ml of cells. The cells wereelectropulsed at 2.0 kV, 25 μF, and 400 ohms. Following electroporation,1 ml SOC (2% Bacto™ Tryptone (Difco, Detroit, Mich.), 0.5% yeast extract(Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mM MgSO₄, 20 mMglucose) was added and then the cells were plated in a 50 μl and 200 μlaliquot on two LB AMP plates (LB broth (Lennox), 1.8% Bacto™ Agar(Difco), 100 mg/L Ampicillin).

The inserts of three clones for each construct were subjected tosequence analysis and one clone for each construct, containing thecorrect sequence, was selected. Larger scale plasmid DNA was isolatedusing a commercially available kit (QIAGEN Plasmid Mega Kit, Qiagen,Valencia, Calif.) according to manufacturer's instructions. The correctconstructs were designated zcyto20/pZMP21 and zcyto21/pZMP21.

Example 2 Expression of Mammalian Constructs in CHO Cells

200 μg of a zcyto20/pZMP21 and zcyto21/pZMP21 construct were digestedwith 200 units of Pvu I at 37° C. for three hours and then wereprecipitated with IPA and spun down in a 1.5 mL microfuge tube. Thesupernatant was decanted off the pellet, and the pellet was washed with1 mL of 70% ethanol and allowed to incubate for 5 minutes at roomtemperature. The tube was spun in a microfuge for 10 minutes at 14,000RPM and the supernatant was aspirated off the pellet. The pellet wasthen resuspended in 750 μl of PF-CHO media in a sterile environment, andallowed to incubate at 60° C. for 30 minutes. CHO cells were spun downand resuspended using the DNA-media solution. The DNA/cell mixture wasplaced in a 0.4 cm gap cuvette and electroporated using the followingparameters: 950 μF, high capacitance, and 300 V. The contents of thecuvette were then removed and diluted to 25 mLs with PF-CHO media andplaced in a 125 mL shake flask. The flask was placed in an incubator ona shaker at 37° C., 6% CO₂, and shaking at 120 RPM.

Example 3 Purification and Analysis of zcyto20-CHO Protein

Purification of Zcyto20-CHO Protein

Recombinant zcyto20 (IL-28A) protein was produced from a pool ofDXB11-CHO cell lines. Cultures were harvested, and the media weresterile filtered using a 0.2 μm filter.

The purification of zcyto20-CHO protein was achieved by the sequentialuse of a Poros HS 50 column (Applied Biosystems, Framingham, Mass.), aMonolithic WCX column (Isco, Inc., Lincoln, Nebr.), a ToyoPearl Butyl650S column (TosoH, Montgomeryville, Pa.), and a Superdex 75 column(Amersham Biosciences, Piscataway, N.J.). Culture media from DXB111-CHOwere adjusted to pH 6.0 before loading onto a Poros 50 HS column. Thecolumn was washed with 50 mM MES (2-Morpholinoethanesulfonic acid), 100mM NaCl, pH 6 and the bound protein was eluted with a 10 column volumes(CV) linear gradient to 60% of 50 mM MES, 2 M NaCl, pH 6. The elutingfractions were collected and the presence of zcyto20 protein wasconfirmed by SDS-PAGE with a Coomassie staining. This fractionscontaining zcyto20 protein were pooled, diluted with double distilledwater to a conductivity of about 20 mS, and loaded onto a Monolithic WCXcolumn. The column was washed with 93% of 50 mM MES, 100 mM NaCl, pH 6,and 7% of 50 mM MES, 2 M NaCl, pH 6. The bound protein was eluted with a25-CV linear gradient from 7% to 50% of 50 mM MES, 2 M NaCl, pH 6. Theeluting fractions were collected and the presence of zcyto20 protein wasconfirmed by SDS-PAGE with a Coomassie staining The fractions containingzcyto20 protein were pooled, adjusted to 1 M ammonium sulfate and loadedonto a ToyoPearl Butyl 650S column. Zcyto20 was eluted with a decreasingammonium sulfate gradient and the fractions containing the pure zcyto20were pooled and concentrated for injection into a Superdex 75 column.Fractions containing zcyto20 protein from the gel filtration column waspooled, concentrated, filtered through a 0.2 μm filter and frozen at−80° C. The concentration of the final purified protein was determinedby a BCA assay (Pierce Chemical Co., Rockford, Ill.) and HPLC-amino acidanalysis.

SDS-PAGE and Western Blotting Analysis of zcyto20-CHO Protein

Recombinant zcyto20 protein was analyzed by SDS-PAGE (Nupage 4-12%Bis-Tris, Invitrogen, Carlsbad, Calif.) and Western blot using rabbitanti-zcyto21-CEE-BV IgG as the primary antibody that cross-reacts tozcyto20-CHO protein. The gel was electrophoresed using Invitrogen'sXcell II mini-cell (Carlsbad, Calif.) and transferred to a 0.2 μmnitrocellulose membrane (Bio-Rad Laboratories, Hercules, Calif.) usingInvitrogen's Xcell II blot module according to directions provided inthe instrument manual. The transfer was run at 500 mA for 50 minutes ina buffer containing 25 mM Tris base, 200 mM glycine, and 20% methanol.The membrane was blocked with 10% non-fat dry milk in 1×PBS for 10minutes then probed with the primary antibody in 1×PBS containing 2.5%non-fat dry milk. The blot was labeled for one hour at room temperaturewhile shaking. For the secondary antibody labeling, blot was washedthree times for 10 minutes each with PBS and then probed with goatanti-rabbit IgG-HRP (Pierce Chemical Co., Rockford, Ill.) for one hour.The blot was washed three times with 1×PBS for 10 minutes each anddeveloped using a 1:1 mixture of SuperSignal® ULTRA reagents (PierceChemical Co., Rockford, Ill.) and the signal was captured using aLumi-Imager (Boehringer Mannheim GmbH, Germany).

Summary of Protein Purification and Analysis

The purified zcyto20 protein from the CHO media migrated predominantlyas a doublet at approximately 20 kDa and a minor triplet dimer at about38 kDa on a 4-12% Bis-Tris gel under non-reducing conditions. They allcollapsed into a single 20 kDa band under reducing conditions. MSpeptide mapping indicated a mixture of two isomers with respect todisulfide linkage and the presence of O-linked glycosylation site.

Example 4 Purification and Analysis of zcyto21-CHO Protein

Purification of Zcyto21-CHO Protein

Recombinant zcyto21 was produced from stable DXB11-CHO cell lines.Cultures were harvested, and the media were sterile filtered using a 0.2μm filter. Proteins were purified from the conditioned media by startingwith a combination of cationic and anionic exchange chromatographyfollowed by a hydrophobic interaction chromatography and a sizeexclusion chromatography. DXB111-CHO culture media were adjusted to pH6.0 before loading onto a Poros 50 HS column (Applied Biosystems,Framingham, Mass.). The column was washed with 1×PBS, pH 6 and the boundprotein was eluted with 5×PBS, pH 8.4. The eluting fraction wascollected and the presence of zcyto21 protein was confirmed by SDS-PAGEwith a Coomassie stain. This fraction was then diluted to a conductivityof 13 mS and its pH adjusted to 8.4 and flowed through a Poros 50 HQcolumn (Applied Biosystems, Framingham, Mass.). The flow-throughcontaining zcyto21 protein were then adjusted to about 127 mS withammonium sulfate and loaded onto a Toyopearl Phenyl 650S column (TosoH,Montgomeryville, Pa.). Zcyto21 protein was eluted with a decreasingammonium sulfate gradient and the fractions containing the pure zcyto21were pooled and concentrated for injection into a Superdex 75 column(Amersham Biosciences, Piscataway, N.J.). The concentration of the finalpurified protein was determined by a BCA assay (Pierce Chemical Co.,Rockford, Ill.) and HPLC-amino acid analysis.

SDS-PAGE and Western Blotting Analysis of zcyto2′-CHO Protein

Recombinant zcyto21 protein was analyzed by SDS-PAGE (Nupage 4-12%Bis-Tris, Invitrogen, Carlsbad, Calif.) and Western blot using rabbitanti-zcyto21-CEE-BV IgG as the primary antibody. The gel waselectrophoresed using Invitrogen's Xcell II mini-cell (Carlsbad, Calif.)and transferred to a 0.2 μm nitrocellulose membrane (Bio-RadLaboratories, Hercules, Calif.) using Invitrogen's Xcell II blot moduleaccording to directions provided in the instrument manual. The transferwas run at 500 mA for 50 minutes in a buffer containing 25 mM Tris base,200 mM glycine, and 20% methanol. The transferred blot was blocked with10% non-fat dry milk in 1×PBS for 10 minutes then probed with theprimary antibody in 1×PBS containing 2.5% non-fat dry milk. The blot waslabeled for one hour at room temperature while shaking. For thesecondary antibody labeling, blot was washed three times for 10 minuteseach with PBS and then probed with goat anti-rabbit IgG-HRP (PierceChemical Co., Rockford, Ill.) for one hour. The blot was washed threetimes with 1×PBS for 10 minutes each and developed using a 1:1 mixtureof SuperSignal® ULTRA reagents (Pierce Chemical Co., Rockford, Ill.) andthe signal was captured using a Lumi-Imager (Boehringer Mannheim GmbH,Germany).

Summary of Protein Purification and Analysis

The purified zcyto21 protein from the CHO media migrated as two or moreapproximately 28 kDa bands on a 4-12% Bis-Tris gel under both reducingand non-reducing conditions. MS peptide mapping indicated a mixture oftwo isomers with respect to disulfide linkage and the presence of oneN-linked glycosylation and several O-linked glycosylation sites.

Example 5 Identification of IL-29 Forms

Peak fractions from purified pools of IL-29 were digested overnight at37° C. with sequencing grade trypsin (Roche Applied Science,Indianapolis, Ind.) in phosphate buffer at approximately pH 6.3 to limitdisulfide re-arrangement. Each digest was analyzed by reversed-phaseHPLC (Agilent, Palo Alto, Calif.) connected in-line to a quadrupole-timeof flight hybrid mass spectrometer (Micromass, Milford Mass.). Spectrawere collected, converted from mass to charge ratio to mass, andcompared to all theoretical peptides and disulfide-linked peptidecombinations resulting from trypsin digestion of IL-29. Disulfides wereassigned by comparing spectra before and after reduction with assignmentof appropriate masses to disulfide linked peptides in IL-29. Thematerial from fraction #20 showed the disulfide pattern C15-C112 andC49-C145 with C171 observed as a S-glutathionyl cysteine (all referringto SEQ ID NO: 4). The material from fraction #51 showed the disulfidepattern C49-C145 and C112-C171 with C15 observed as an S-glutathionylcysteine (referring to SEQ ID NO:4).

Example 6 E. coli Expression Plasmids

Construction of Expression Vector, pTAP237

Plasmid pTAP237 was generated by inserting a PCR-generated linker intothe SmaI site of pTAP186 by homologous recombination. Plasmid pTAP186was derived from the plasmids pRS316 (a Saccharomyces cerevisiae shuttlevector) and pMAL-c2, an E. coli expression plasmid derived from pKK223-3and comprising the tac promoter and the rrnB terminator. Plasmid pTAP186contains a kanamycin resistance gene in which the Sma I site has beendestroyed and has NotI and SfiI sites flanking the yeast ARS-CEN6 andURA3 sequences, facilitating their removal from the plasmid by digestionwith Nod. The PCR-generated linker replaced the expression couplersequence in pTAP186 with the synthetic RBS II sequence. It was preparedfrom 100 pmoles each of oligonucleotides zc29,740 and zc29,741, as shownin SEQ ID NOS: 44 and 45, respectively, and approximately 5 pmoles eachof oligonucleotides zc29,736 and zc29,738, as shown in SEQ ID NOS: 46and 47, respectively. These oligonucleotides were combined by PCR forten cycles of 94° C. for 30 seconds, 50° C. for 30 seconds, and 72° C.for 30 seconds, followed by 4° C. soak. The resulting PCR products wereconcentrated by precipitation with two times the volume of 100% ethanol.Pellet was resuspended in 10 μL water to be used for recombining intothe recipient vector pTAP186 digested with SmaI to produce the constructcontaining the synthetic RBS II sequence. Approximately 1 μg of thePCR-generated linker and 100 ng of pTAP186 digested with SmaI were mixedtogether and transformed into competent yeast cells (S. cerevisiae). Theyeast was then plated onto -URA D plates and left at room temperaturefor about 72 hours. Then the Ura+transformants from a single plate wereresuspended in 1 mL H₂O and spun briefly to pellet the yeast cells. Thecell pellet was resuspended in 0.5 mL of lysis buffer. DNA was recoveredand transformed into E. coli MC1061. Clones were screened by colony PCRas disclosed above using 20 pmoles each of oligonucleotides zc29,740 andzc29,741, as shown in SEQ ID NOS: 44 and 45, respectively. Clonesdisplaying the correct size band on an agarose gel were subject tosequence analysis. The correct plasmid was designated pTAP237.

Example 7 Codon Optimization of IL-29 Cysteine Mutant

Codon Optimization Generation of the IL-29 Wildtype Expression Construct

Native human IL-29 gene sequence was not well expressed in E. colistrain W3110. Examination of the codons used in the IL-29 codingsequence indicated that it contained an excess of the least frequentlyused codons in E. coli with a CAI value equal to 0.206. The CAI is astatistical measure of synonymous codon bias and can be used to predictthe level of protein production (Sharp et al., Nucleic Acids Res.15(3):1281-95, 1987). Genes coding for highly expressed proteins tend tohave high CAI values (>0.6), while proteins encoded by genes with lowCAI values (≦0.2) are generally inefficiently expressed. This suggesteda reason for the poor production of IL-29 in E. coli. Additionally, therare codons are clustered in the second half of the message leading tohigher probability of translational stalling, premature termination oftranslation, and amino acid misincorporation (Kane J F. Curr. Opin.Biotechnol. 6(5):494-500, 1995).

It has been shown that the expression level of proteins whose genescontain rare codons can be dramatically improved when the level ofcertain rare tRNAs is increased within the host (Zdanovsky et al.,Applied Enviromental Microb. 66:3166-3173, 2000; You et al.,Biotechniques 27:950-954, 1999). The pRARE plasmid carries genesencoding the tRNAs for several codons that are rarely used E. coli(argU, argW, leuW, proL, ileX and glyT). The genes are under the controlof their native promoters (Novy, ibid.) Co-expression with pRAREenhanced IL-29 production in E. coli and yield approximately 200 mg/L.These data suggest that re-resynthesizing the gene coding for IL-29 withmore appropriate codon usage provides an improved vector for expressionof large amounts of IL-29.

The codon optimized IL-29 coding sequence was constructed from sixteenoverlapping oligonucleotides: zc44,566 (SEQ ID NO:48), zc44,565 (SEQ IDNO:49), zc44,564 (SEQ ID NO:50), zc44,563 (SEQ ID NO:51), zc44,562 (SEQID NO:52), zc44,561 (SEQ ID NO:53), zc44,560 (SEQ ID NO:54), zc244,559(SEQ ID NO:55), zc44,558 (SEQ ID NO:56), zc44,557 (SEQ ID NO:57). Primerextension of these overlapping oligonucleotides followed by PCRamplication produced a full length IL-29 gene with codons optimized forexpression in E. coli. The final PCR product was inserted intoexpression vector pTAP237 by yeast homologous recombination. Theexpression construct was extracted from yeast and transformed intocompetent E. coli MC1061. Clones resistance to kanamycin were identifiedby colony PCR. A positive clone was verified by sequencing andsubsequently transformed into production host strain W3110. Theexpression vector with the optimized IL-29 sequence was named pSDH184.The resulting gene was expressed very well in E. coli. expression levelswith the new construct increased to around 250 mg/L.

Generation of the Codon Optimized zcyto21 C172S Cysteine MutantExpression Construct

The strategy used to generate the zcyto21 C172S Cysteine mutant is basedon the QuikChange Site-Directed Mutagenesis Kit (Stratagene). Primerswere designed to introduce the C172S mutation based on manufacturer'ssuggestions. These primers were designated ZG44,340 (SEQ ID NO: 58) andZG44,341 (SEQ ID NO: 59). PCR was performed to generate the zcyto21C172S Cysteine mutant according to QuikChange Mutagenesis instructions.Five identical 50 μl reactions were set-up. 2.5 μl pSDH175 (missingyeast vector backbone sequence) DNA was used as template per reaction. APCR cocktail was made up using the following amounts of reagents: 30 μl10×PCR buffer, 125 ng (27.42 μl) ZG44,340, 125 ng (9.18 μl) ZG44,341, 6μl dNTP, 6 μl Pfu Turbo polymerase (Stratagene, La Jolla, Calif.), and206.4 μl water. 47.5 μl of the cocktail was aliquoted into eachreaction. The PCR conditions were as follows: 1 cycle of 95° C. for 30seconds followed by 16 cycles of 95° C. for 30 seconds, 55° C. for 1minute, 68° C. for 7 minutes, followed by 1 cycle at 68° C. for 7minutes, and ending with a 4° C. hold. All five PCR reactions wereconsolidated into one tube. As per manufacturer's instructions, 5 μlDpnI restriction enzyme was added to the PCR reaction and incubated at37° C. for 2 hours. DNA was precipitated my adding 10% 3 Molar SodiumAcetate and two volumes of 100% ethanol. Precipitation was carried-outat −20° C. for 20 minutes. DNA was spun at 14,000 rpm for 5 minutes andpellet was speed-vac dried. DNA pellet was resuspended in 20 μl water.DNA resulting from PCR was transformed into E. coli strain DH10B. 5 μlDNA was mixed with 40 μl ElectroMAX DH10B cells (Invitrogen). Cells andDNA mixture were then electroporated in a 0.1 cm cuvette (Bio-Rad) usinga Bio-Rad Gene Pulser II™ set to 1.75 kV, 100Ω, and 25 μF.Electroporated cells were then outgrown at 37° C. for 1 hour. Mixturewas plated on an LB+25 μg/ml kanamycin plate and incubated at 37° C.overnight. Ten clones were screened for presence of zcyto21 C172Sinsert. DNA was isolated from all ten clones using the QIAprep™ SpinMiniprep Kit (Qiagen, Valencia, Calif.) and analyzed for presence ofinsert by cutting with XbaI and PstI restriction enzymes. Nine clonescontained insert and were sequenced to insure the zcyto21 C172S mutationhad been introduced. A clone was sequence verified and was subsequentlylabeled pSDH188.

Example 8 E. coli IL-29 Expression Construct

A DNA fragment of IL-29 containing the wildtype sequence was isolatedusing PCR. Primers zc41,212 (SEQ ID NO: 60) containing 41 base pair (bp)of vector flanking sequence and 24 by corresponding to the aminoterminus of IL-29, and primer zc41,041 (SEQ ID NO: 61) contained 38 bpcorresponding to the 3′ end of the vector which contained the zcyto21insert were used in the reaction. The PCR conditions were as follows: 25cycles of 94° C. for 30 seconds, 50° C. for 30 seconds, and 72° C. for 1minute; followed by a 4° C. soak. A small sample (2-4 μL) of the PCRsample was run on a 1% agarose gel with 1×TBE buffer for analysis, andthe expected band of approximately 500 bp fragment was seen. Theremaining volume of the 100 μL reaction was precipitated with 200 μLabsolute ethanol. The pellet was resuspended in 10 μL water to be usedfor recombining into recipient vector pTAP238 cut with SmaI to producethe construct encoding the zcyto21 as disclosed above. The clone withcorrect sequence was designated as pTAP377. Clone pTAP377 was digestedwith Not1/Nco1 (10 μl DNA, 5 μl buffer 3 New England BioLabs, 2 μL Not1, 2 μL Nco1, 31 μL water for 1 hour at 37° C.) and religated with T4DNA ligase buffer (7 μL of the previous digest, 2 μL of 5× buffer, 1 μLof T4 DNA ligase). This step removed the yeast sequence, CEN-ARS, tostreamline the vector. The pTAP337 DNA was diagnostically digested withPvu2 and Pst1 to confirm the absence of the yeast sequence. P/taP377 DNAwas transformed into E. coli strain W3110/pRARE, host strain carryingextra copies of rare E. coli tRNA genes.

Example 9 E. coli IL-28A Expression Construct

A DNA fragment containing the wildtype sequence of zcyto20 (as shown inSEQ ID NO: 1) was isolated using PCR. Primers zc43,431 (SEQ ID NO: 62)containing 41 bp of vector flanking sequence and 24 bp corresponding tothe amino terminus of zcyto20, and primer zc43,437 (SEQ ID NO: 63)contained 38 bp corresponding to the 3′ end of the vector whichcontained the zcyto20 insert. The PCR conditions were as follows: 25cycles of 94° C. for 30 seconds, 50° C. for 30 seconds, and 72° C. for 1minute; followed by a 4° C. soak. A small sample (2-4 μL) of the PCRsample was run on a 1% agarose gel with 1×TBE buffer for analysis, andthe expected band of approximately 500 bp fragment was seen. Theremaining volume of the 100 μL reaction was precipitated with 200 μLabsolute ethanol. The pellet was resuspended in 10 μL water to be usedfor recombining into recipient vector pTAP238 cut with SmaI to producethe construct encoding the zcyto20 as disclosed above. The clone withcorrect sequence was designated as pYEL7. It was digested with Not1/Nco1(10 μl DNA, 5 μl buffer 3 New England BioLabs, 2 μL Not1, 2 μL Nco1, 31μL water for 1 hour at 37° C.) and religated with T4 DNA ligase buffer(7 μL of the previous digest, 2 μL of 5× buffer, 1 μL of T4 DNA ligase).This step removed the yeast sequence, CEN-ARS, to streamline the vector.The relegated pYEL7 DNA was diagnostically digested with Pvu2 and Pst1to confirm the absence of the yeast sequence. PYEL7 DNA was transformedinto E. coli strain W3110/pRARE.

Example 10 zcyto21 C172S Cysteine Mutant Expression Construct

The strategy used to generate the zcyto21 C172S Cysteine mutant (SEQ IDNO: 28) is based on the QuikChange® Site-Directed Mutagenesis Kit(Stratagene, La Jolla, Calif.). Primers were designed to introduce theC172S mutation based on manufacturer's suggestions. These primers weredesignated ZG44,327 and ZG44,328 (SEQ ID NOS: 64 and 65, respectively).PCR was performed to generate the zcyto21 C172S Cysteine mutantaccording to QuikChange Mutagenesis instructions. Five identical 50 μlreactions were set-up. 2.5 μl pTAP377 (missing yeast vector backbonesequence) DNA was used as template per reaction. A PCR cocktail was madeup using the following amounts of reagents: 30 μl 10×PCR buffer, 125 ng(27.42 μl) ZG44,327 (SEQ ID NO: 64), 125 ng (9.18 μl) ZG44,328 (SEQ IDNO: 65), 6 μl dNTP, 6 μl Pfu Turbo polymerase (Strategene), and 206.4 μlwater. 47.5 μl of the cocktail was aliquoted into each reaction. The PCRconditions were as follows: 1 cycle of 95° C. for 30 seconds followed by16 cycles of 95° C. for 30 seconds, 55° C. for 1 minute, 68° C. for 7minutes, followed by 1 cycle at 68° C. for 7 minutes, and ending with a4° C. hold. All five PCR reactions were consolidated into one tube. Asper manufacturer's instructions, 5 μl DpnI restriction enzyme was addedto the PCR reaction and incubated at 37° C. for 2 hours. DNA wasprecipitated my adding 10% 3 Molar Sodium Acetate and two volumes of100% ethanol (Aaper Alcohol, Shelbyville, Ky.). Precipitation wascarried-out at −20° C. for 20 minutes. DNA was spun at 14,000 rpm for 5minutes and pellet was speed-vac dried. DNA pellet was resuspended in 20μl water. DNA resulting from PCR was transformed into E. coli strainDH10B. 5 μl DNA was mixed with 40 μl ElectroMAX DH10B cells (Invitrogen,Carlsbad, Calif.). Cells and DNA mixture were then electroporated in a0.1 cm cuvette (Bio-Rad, Hercules, Calif.) using a Bio-Rad Gene PulserII™ set to 1.75 kV, 10052, and 25 μF. Electroporated cells were thenoutgrown at 37° C. for 1 hour. Mixture was plated on an LB+25 μg/mlkanamycin plate and incubated at 37° C. overnight. Ten clones werescreened for presence of IL-29 insert. DNA was isolated from all tenclones using the QIAprep™ Spin Miniprep Kit (Qiagen) and analyzed forpresence of insert by cutting with XbaI (Roche) and PstI (New EnglandBiolabs) restriction enzymes. Nine clones contained insert and weresequenced to insure the zcyto21 C172S mutation had been introduced. Aclone (isolet #6) was sequence verified and was subsequently labeledpSDH171. A similar strategy can be implemented to generate a zcyto21C15S mutant.

Example 11 zcyto20 C49S Cysteine Mutant Expression Construct

The zcyto20 C49S Cysteine mutant coding sequence was generated byoverlap PCR (SEQ ID NO: 20). The first 187 bases of the wildtype IL-28Asequence (SEQ ID NO:1) was generated by PCR amplification using pYEL7(SEQ ID NO: 67) as template and oligonucleotide primers zc43,431 (SEQ IDNO: 62) and zc45,399 (SEQ ID NO: 66). The second DNA fragment from base105 to 531 was generated by PCR amplification using pYEL7 (SEQ ID NO:67) as template and oligonucleotide primers zc45,398 (SEQ ID NO: 68) andzc43,437 (SEQ ID NO: 63). Primers zc45,399 (SEQ ID NO: 66) and zc45,398(SEQ ID NO: 68) contained the specific modified sequence which changedthe cysteine 49 to a serine. These two PCR products were combined andPCR overlap amplified using oligonucleotide primers zc43,431 (SEQ ID NO:62) and zc43,437 (SEQ ID NO: 63). The final PCR product was insertedinto expression vector pTAP238 by yeast homologous recombination(Raymond et al. Biotechniques. Jan. 26(1):134-8, 140-1, 1999). Theexpression construct was extracted from yeast and transformed intocompetent E. coli DH10B. Kanamycin resistant clones were screened bycolony PCR. A positive clone was verified by sequencing and subsequentlytransformed into production host strain W3110/pRARE. The expressionconstruct with the zcyto20 C49S Cysteine mutant coding sequence wasnamed pCHAN9.

Example 12

zcyto20 C51S Cysteine Mutant Expression Construct

The zcyto20 C51S Cysteine mutant coding sequence was generated byoverlap PCR (SEQ ID NO: 24). The first 193 bases of the wildtype IL-28Asequence was generated by PCR amplification using pYEL7 (SEQ ID NO: 67)as template and oligonucleotide primers zc43,431 (SEQ ID NO: 62) andzc45,397 (SEQ ID NO: 63). The second DNA fragment from base 111 to 531was generated by PCR amplification using pYEL7 (SEQ ID NO: 67) astemplate and oligonucleotide primers zc45,396 (SEQ ID NO:70) andzc43,437 (SEQ ID NO: 63). Primers zc45,397 (SEQ ID NO: 69) and zc45,396(SEQ ID NO: 70) contained the specific modified sequence which changedthe cysteine51 to a serine. These two PCR products were combined and PCRoverlap amplified using oligonucleotide primers zc43,431 (SEQ ID NO: 62)and zc43,437 (SEQ ID NO: 63). The final PCR product was inserted intoour in-house expression vector pTAP238 by yeast homologous recombination(Raymond et al. supra). The expression construct was extracted fromyeast and transformed into competent E. coli DH10B. Kanamycin resistantclones were screened by colony PCR. A positive clone was verified bysequencing and subsequently transformed into production host strainW3110/pRARE. The expression construct with the zcyto20 C50S Cysteinemutant coding sequence was named pCHAN10.

Example 13 Expression of Il-28A, IL-29 and Cys to Ser Cysteine Mutantsin E. coli

In separate experiments, E. coli transformed with each of the expressionvectors described in Examples 6-9 were inoculated into 100 mL SuperbrothII medium (Becton Dickinson, San Diego, Calif.) with 0.01% Antifoam 289(Sigma Aldrich, St. Louis, Mo.), 30 μg/ml kanamycin, 35 μg/mlchloramphenicol and cultured overnight at 37° C. A 5 mL inoculum wasadded to 500 mL of same medium in a 2 L culture flask which was shakenat 250 rpm at 37° C. until the culture attained an OD600 of 4. IPTG wasthen added to a final concentration of 1 mM and shaking was continuedfor another 2.5 hours. The cells were centrifuged at 4,000×g for 10 minat 4° C. The cell pellets were frozen at −80° C. until use at a latertime.

Example 14 Refolding and Purification of IL-28

Inclusion Body Preparation

Human wildtype IL-29 was expressed in E. coli strain W3110 as inclusionbodies as described above. A cell pellet from a fed-batch fermentationwas resuspended in 50 mM Tris, pH 7.3. The suspension was passed throughan APV-Gaulin homogenizer (Invensys APV, Tonawanda, New York) threetimes at 8000 psi. The insoluble material was recovered bycentrifugation at 15,000 g for 30 minutes. The pellet was washedconsecutively with 50 mM Tris, 1% (v/v) Triton X100, pH 7.3 and 4 MUrea. The inclusion body was then dispersed in 50 mM Tris, 6 M guanidinehydrochloride, 5 mM DTT at room temperature for 1 hour. The material wasthen centrifuged at 15,000 g for 1 hour. The supernatant from this stepcontains reduced soluble IL-29.

Refolding

The solubilized IL-29 was diluted slowly into 50 mM Tris, pH 8, 0.75 MArginine, 0.05% PEG3350, 2 mM MgCl₂, 2 mM CaCl₂, 0.4 mM KCl, 10 mM NaCl,4 mM reduced Glutathione, 0.8 mM oxidized Glutathione at roomtemperature while stirring. The final concentration of IL-29 in therefolding buffer was 0.1 mg/ml. The refolding mixture was left at roomtemperature overnight. Concentrated acetic acid was then used to adjustthe pH of the suspension to 5. The suspension was then filtered througha 0.2 μm filter. RP-HPLC analysis of the refolding mixture showed twoprominent peaks.

Purification

The refolding mixture was in-line diluted (1:2) with 50 mM NaOAc at pH 5and loaded onto a Pharmacia SP Sepharose Fast Flow cation exchangecolumn (North Peapack, N.J.). The column was washed with 3 columnvolumes of 50 mM NaOAc, 400 mM NaCl, pH 5. The bound IL-29 was elutedwith 50 mM NaOAc, 1.4 M NaCl, pH 5. Solid (NH₄)₂SO₄ was added to theelute pool of the cation exchange step so that the final concentrationof (NH₄)₂SO₄ was 0.5 M. The material was then loaded onto a ToyoPearlPhenyl 650S HIC column (Tosoh Biosep, Montgomery, Pa.). The column wasthen washed with 3 column volumes of 50 mM NaOAc, 1 M (NH₄)₂SO₄, pH 5. Alinear gradient of 10 column volumes from 50 mM NaOAc, 1 M (NH₄)₂SO₄, pH5 to 50 mM NaOAc, pH 5 was used to elute the bound zcyto21. Fractionswere collected of the elute. Two prominent peaks were observed in thisstep. RP-HPLC analysis of the elute fractions was performed. Twoproducts corresponding to two disulfide bond isomers were produced afterfinal buffer exchange into PBS, pH 7.3.

Example 15 Refolding and Purification of IL-29 Cysteine Mutant

As described in Example 3, purification of IL-29 produced two disulfidebond isomers. A HIC FPLC step was employed to separate the two forms.The separation was not baseline resolved. Severe “Peak Shaving” had tobe used to obtain substantially pure isomers (>95%). The yield for thisstep and by extension for the whole process suffered. The final yieldswere 8% and 9% for the C15-C112 form and C112-C171 form respectively.Wildtype IL-29 produced in CHO and baculovirus (BV) systems also showedsimilar phenomena. It was established that the C15-C112 form of theisomer is homologous in disulfide bond patterns to type I INF's. TheC15-C112 form also demonstrated 30-fold higher bioactivity than theC112-C171 form in an ISRE assay (see below).

Refolding and Purification of zcyto21 Cys172Ser Mutein

The inclusion body preparation, refolding and purification of zcyto21C172S polypeptide (SEQ ID NO:29) is essentially the same as those ofIL-29 wild-type (SEQ ID NO:4). RP-HPLC analysis of the refolding mixtureof the mutein showed only one prominent peak corresponding to theC15-C112 form of the wild-type IL-29. Subsequent HIC chromatography showonly a single peak. It was therefore unnecessary to employ severe “peakshaving”. The final yield for the entire process is close to 50%. Thezcyto21 Cys172Ser polypeptide (SEQ ID NO:29) showed equivalentbioactivity to the C15-C112 form of wild-type IL-29 in ISRE assay shownin Example 16.

Example 16 IL-28RA mRNA Expression in Liver and Lymphocyte Subsets

In order to further examine the mRNA distribution for IL-28RA,semi-quantitative RT-PCR was performed using the SDS 7900HT system(Applied Biosystems, CA). One-step RT-PCR was performed using 100 ngtotal RNA for each sample and gene-specific primers. A standard curvewas generated for each primer set using Bjab RNA and all sample valueswere normalized to HPRT. The normalized values for IFNAR2 and CRF2-4 arealso shown.

Table 7: B and T cells express significant levels of IL-28RA mRNA. Lowlevels are seen in dendritic cells and most monocytes.

TABLE 7 Cell/Tissue IL-28RA IFNAR2 CRF2-4 Dendritic Cells unstim .04 5.99.8 Dendritic Cells + IFNg .07 3.6 4.3 Dendritic Cells .16 7.85 3.9CD14+ stim'd with LPS/IFNg .13 12 27 CD14+ monocytes resting .12 11 15.4Hu CD14+ Unact. 4.2 TBD TBD Hu CD14+ 1 ug/ml LPS act. 2.3 TBD TBD H.Inflamed tonsil 3 12.4 9.5 H. B-cells + PMA/Iono 4 & 24 hrs 3.6 1.3 1.4Hu CD19+ resting 6.2 TBD TBD Hu CD19+ 4 hr. PMA/Iono 10.6 TBD TBD HuCD19+ 24 hr Act. PMA/Iono 3.7 TBD TBD IgD+ B-cells 6.47 13.15 6.42 IgM+B-cells 9.06 15.4 2.18 IgD− B-cells 5.66 2.86 6.76 NKCells + PMA/Iono 06.7 2.9 Hu CD3+ Unactivated 2.1 TBD TBD CD4+ resting .9 8.5 29.1 CD4+Unstim 18 hrs 1.6 8.4 13.2 CD4+ + Poly I/C 2.2 4.5 5.1 CD4+ + PMA/Iono.3 1.8 .9 CD3 neg resting 1.6 7.3 46 CD3 neg unstim 18 hrs 2.4 13.2 16.8CD3 neg + Poly I/C 18 hrs 5.7 7 30.2 CD3 neg + LPS 18 hrs 3.1 11.9 28.2CD8+ unstim 18 hrs 1.8 4.9 13.1 CD8+ stim'd with PMA/Ion 18 hrs .3 .61.1As shown in Table 8, normal liver tissue and liver derived cell linesdisplay substantial levels of IL-28RA and CRF2-4 mRNA.

TABLE 8 IL- Cell/Tissue 28RA IFNAR2 CRF2-4 HepG2 1.6 3.56 2.1 HepG2 UGAR5/10/02 1.1 1.2 2.7 HepG2, CGAT HKES081501C 4.3 2.1 6 HuH7 5/10/02 1.6316 2 HuH7 hepatoma—CGAT 4.2 7.2 3.1 Liver, normal—CGAT #HXYZ020801K 11.73.2 8.4 Liver, NAT—Normal adjacent tissue 4.5 4.9 7.7 Liver, NAT—Normaladjacent tissue 2.2 6.3 10.4 Hep SMVC hep vein 0 1.4 6.5 Hep SMCA hep.Artery 0 2.1 7.5 Hep. Fibro 0 2.9 6.2 Hep. Ca. 3.8 2.9 5.8 Adenoca liver8.3 4.2 10.5 SK-Hep-1 adenoca. Liver .1 1.3 2.5 AsPC-1 Hu. Pancreaticadenocarc. .7 .8 1.3 Hu. Hep. Stellate cells .025 4.4 9.7

As shown in Table 9, primary airway epithelial cells contain abundantlevels of IL-28RA and CRF2-4.

TABLE 9 Cell/Tissue IL-28RA IFNAR2 CRF2-4 U87MG - glioma 0 .66 .99 NHBEunstim 1.9 1.7 8.8 NHBE + TNF-alpha 2.2 5.7 4.6 NHBE + poly I/C 1.8 ndnd Small Airway Epithelial Cells 3.9 3.3 27.8 NHLF—Normal human lungfibroblasts 0 nd nd

As shown in Table 10, IL-28RA is present in normal and diseased liverspecimens, with increased expression in tissue from Hepatitis C andHepatitis B infected specimens.

TABLE 10 Cell/Tissue IL-28RA CRF2-4 IFNAR2 Liver with CoagulationNecrosis 8.87 15.12 1.72 Liver with Autoimmune Hepatitis 6.46 8.90 3.07Neonatal Hepatitis 6.29 12.46 6.16 Endstage Liver disease 4.79 17.0510.58 Fulminant Liver Failure 1.90 14.20 7.69 Fulminant Liver failure2.52 11.25 8.84 Cirrhosis, primary biliary 4.64 12.03 3.62 CirrhosisAlcoholic (Laennec's) 4.17 8.30 4.14 Cirrhosis, Cryptogenic 4.84 7.135.06 Hepatitis C+, with cirrhosis 3.64 7.99 6.62 Hepatitis C+ 6.32 11.297.43 Fulminant hepatitis secondary to Hep A 8.94 21.63 8.48 Hepatitis C+7.69 15.88 8.05 Hepatitis B+ 1.61 12.79 6.93 Normal Liver 8.76 5.42 3.78Normal Liver 1.46 4.13 4.83 Liver NAT 3.61 5.43 6.42 Liver NAT 1.9710.37 6.31 Hu Fetal Liver 1.07 4.87 3.98 Hepatocellular Carcinoma 3.583.80 3.22 Adenocarcinoma Liver 8.30 10.48 4.17 hep. SMVC, hep. Vein 0.006.46 1.45 Hep SMCA hep. Artery 0.00 7.55 2.10 Hep. Fibroblast 0.00 6.202.94 HuH7 hepatoma 4.20 3.05 7.24 HepG2 Hepatocellular carcinoma 3.405.98 2.11 SK-Hep-1 adenocar. Liver 0.03 2.53 1.30 HepG2 Unstim 2.06 2.982.28 HepG2 + zcyto21 2.28 3.01 2.53 HepG2 + IFNα 2.61 3.05 3.00 NormalFemale Liver - degraded 1.38 6.45 4.57 Normal Liver - degraded 1.93 4.996.25 Normal Liver - degraded 2.41 2.32 2.75 Disease Liver - degraded2.33 3.00 6.04 Primary Hepatocytes from Clonetics 9.13 7.97 13.30

As shown in Tables 11-15, IL-28RA is detectable in normal B cells, Blymphoma cell lines, T cells, T lymphoma cell lines (Jurkat), normal andtransformed lymphocytes (B cells and T cells) and normal humanmonocytes.

TABLE 11 HPRT IL-28RA IL-28RA IFNR2 CRF2-4 Mean Mean norm IFNAR2 normCRF2-4 Norm CD14+ 24 hr unstim #A38 13.1 68.9 5.2 92.3 7.0 199.8 15.2CD14+ 24 hr stim #A38 6.9 7.6 1.1 219.5 31.8 276.6 40.1 CD14+ 24 hrunstim #A112 17.5 40.6 2.3 163.8 9.4 239.7 13.7 CD14+ 24 hr stim #A11211.8 6.4 0.5 264.6 22.4 266.9 22.6 CD14+ rest #X 32.0 164.2 5.1 1279.739.9 699.9 21.8 CD14+ + LPS #X 21.4 40.8 1.9 338.2 15.8 518.0 24.2 CD14+24 hr unstim #A39 26.3 86.8 3.3 297.4 11.3 480.6 18.3 CD14+ 24 hr stim#A39 16.6 12.5 0.8 210.0 12.7 406.4 24.5 HL60 Resting 161.2 0.2 0.0214.2 1.3 264.0 1.6 HL60 + PMA 23.6 2.8 0.1 372.5 15.8 397.5 16.8 U937Resting 246.7 0.0 0.0 449.4 1.8 362.5 1.5 U937 + PMA 222.7 0.0 0.0 379.21.7 475.9 2.1 Jurkat Resting 241.7 103.0 0.4 327.7 1.4 36.1 0.1 JurkatActivated 130.7 143.2 1.1 Colo205 88.8 43.5 0.5 HT-29 26.5 30.5 1.2

TABLE 12 HPRT SD IL-28RA SD Mono 24 hr unstim #A38 0.6 2.4 Mono 24 hrstim #A38 0.7 0.2 Mono 24 hr unstim #A112 2.0 0.7 Mono 24 hr stim #A1120.3 0.1 Mono rest #X 5.7 2.2 Mono + LPS #X 0.5 1.0 Mono 24 hr unstim#A39 0.7 0.8 Mono 24 hr stim #A39 0.1 0.7 HL60 Resting 19.7 0.1 HL60 +PMA 0.7 0.4 U937 Resting 7.4 0.0 U937 + PMA 7.1 0.0 Jurkat Resting 3.71.1 Jurkat Activated 2.4 1.8 Colo205 1.9 0.7 HT-29 2.3 1.7

TABLE 13 Mean Mean Mean IL- Mean Hprt IFNAR2 28RA CRF CD3+/CD4+ 0 10.185.9 9.0 294.6 CD4/CD3+ Unstim 18 hrs 12.9 108.7 20.3 170.4 CD4+/CD3+ +Poly I/C 18 hrs 24.1 108.5 52.1 121.8 CD4+/CD3+ + PMA/Iono 18 hrs 47.883.7 16.5 40.8 CD3 neg 0 15.4 111.7 24.8 706.1 CD3 neg unstim 18 hrs15.7 206.6 37.5 263.0 CD3 neg + Poly I/C 18 hrs 9.6 67.0 54.7 289.5 CD3neg + LPS 18 hrs 14.5 173.2 44.6 409.3 CD8+ Unstim. 18 hrs 6.1 29.7 11.179.9 CD8+ + PMA/Iono 18 hrs 78.4 47.6 26.1 85.5 12.8.1 - NHBE Unstim47.4 81.1 76.5 415.6 12.8.2 - NHBE + TNF-alpha 42.3 238.8 127.7 193.9SAEC 15.3 49.9 63.6 426.0

TABLE 14 IL-28RA CRF IFNAR2 IL-28RA CRF IFNAR2 Norm Norm Norm SD SD SDCD3+/CD4+ 0 0.9 29.1 8.5 0.1 1.6 0.4 CD4/CD3+ Unstim 18 hrs 1.6 13.2 8.40.2 1.6 1.4 CD4+/CD3+ + Poly I/C 18 hrs 2.2 5.1 4.5 0.1 0.3 0.5CD4+/CD3+ + PMA/Iono 18 hrs 0.3 0.9 1.8 0.0 0.1 0.3 CD3 neg 0 1.6 46.07.3 0.2 4.7 1.3 CD3 neg unstim 18 hrs 2.4 16.8 13.2 0.4 2.7 2.3 CD3neg + Poly I/C 18 hrs 5.7 30.2 7.0 0.3 1.7 0.8 CD3 neg + LPS 18 hrs 3.128.2 11.9 0.4 5.4 2.9 CD8+ Unstim. 18 hrs 1.8 13.1 4.9 0.1 1.1 0.3CD8+ + PMA/Iono 18 hrs 0.3 1.1 0.6 0.0 0.1 0.0 12.8.1 - NHBE Unstim 1.68.8 1.7 0.1 0.4 0.1 12.8.2 - NHBE + TNF-alpha 3.0 4.6 5.7 0.1 0.1 0.1SAEC 4.1 27.8 3.3 0.2 1.1 0.3

TABLE 15 SD SD IL- SD Hprt SD IFNAR2 28RA CRF CD3+/CD4+ 0 0.3 3.5 0.612.8 CD4/CD3+ Unstim 18 hrs 1.4 13.7 1.1 8.5 CD4+/CD3+ + Poly I/C 18 hrs1.3 9.8 1.6 3.4 CD4+/CD3+ + PMA/Iono 18 hrs 4.0 10.3 0.7 3.7 CD3 neg 01.4 16.6 1.6 28.6 CD3 neg unstim 18 hrs 2.4 16.2 2.7 12.6 CD3 neg + PolyI/C 18 hrs 0.5 7.0 1.0 8.3 CD3 neg + LPS 18 hrs 1.0 39.8 5.6 73.6 CD8+Unstim. 18 hrs 0.2 1.6 0.5 6.1 CD8+ + PMA/Iono 18 hrs 1.3 1.7 0.2 8.112.8.1 - NHBE Unstim 2.4 5.6 2.7 2.8 12.8.2 - NHBE + TNF-alpha 0.5 3.43.5 3.4 SAEC 0.5 4.8 1.8 9.9

Example 17 Mouse IL-28 Does Not Have Antiproliferative Effect on Mouse BCells

Mouse B cells were isolated from 2 Balb/C spleens (7 months old) bydepleting CD43+ cells using MACS magnetic beads. Purified B cells werecultured in vitro with LPS, anti-IgM or anti-CD40 monoclonal antibodies.Mouse IL-28 or mouse IFNα was added to the cultures and ³H-thymidine wasadded at 48 hrs. and ³H-thymidine incorporation was measured after 72hrs. culture.

IFNα at 10 ng/ml inhibited ³H-thymidine incorporation by mouse B cellsstimulated with either LPS or anti-IgM. However mouse IL-28 did notinhibit ³H-thymidine incorporation at any concentration tested including1000 ng/ml. In contrast, both mIFNα and mouse IL-28 increased ³Hthymidine incorporation by mouse B cells stimulated with anti-CD40 MAb.

These data demonstrate that mouse IL-28 unlike IFNa displays noantiproliferative activity even at high concentrations. In addition,zcyto24 enhances proliferation in the presence of anti-CD40 MAbs. Theresults illustrate that mouse IL-28 differs from IFNα in that mouseIL-28 does not display antiproliferative activity on mouse B cells, evenat high concentrations. In addition, mouse IL-28 enhances proliferationin the presence of anti-CD40 monoclonal antibodies.

Example 18 Bone Marrow Expansion Assay

Fresh human marrow mononuclear cells (Poietic Technologies,Gaithersburg, Md.) were adhered to plastic for 2 hrs in αMEM, 10% FBS,50 micromolar β-mercaptoethanol, 2 ng/ml FLT3L at 37° C. Non adherentcells were then plated at 25,000 to 45,000 cells/well (96 well tissueculture plates) in aMEM, 10% FBS, 50 micromolar β-mercaptoethanol, 2ng/ml FLT3L in the presence or absence of 1000 ng/ml IL-29-CEE, 100ng/ml IL-29-CEE, 10 ng/ml IL-29-CEE, 100 ng/ml IFN-α2a, 10 ng/ml IFN-α2aor 1 ng/ml IFN-α2a. These cells were incubated with a variety ofcytokines to test for expansion or differentiation of hematopoieticcells from the marrow (20 ng/ml IL-2, 2 ng/ml IL-3, 20 ng/ml IL-4, 20ng/ml IL-5, 20 ng/ml IL-7, 20 ng/ml IL-10, 20 ng/ml IL-12, 20 ng/mlIL-15, 10 ng/ml IL-21 or no added cytokine). After 8 to 12 days AlamarBlue (Accumed, Chicago, Ill.) was added at 20 microliters/well. Plateswere further incubated at 37° C., 5% CO, for 24 hours. Plates were readon the Fmax™ plate reader (Molecular Devices Sunnyvale, Calif.) usingthe SoftMax™ Pro program, at wavelengths 544 (Excitation) and 590(Emission). Alamar Blue gives a fluourometric readout based on themetabolic activity of cells, and is thus a direct measurement of cellproliferation in comparison to a negative control.

IFN-α2a caused a significant inhibition of bone marrow expansion underall conditions tested. In contrast, IL-29 had no significant effect onexpansion of bone marrow cells in the presence of IL-3, IL-4, IL-5,IL-7, IL-10, IL-12, IL-21 or no added cytokine. A small inhibition ofbone marrow cell expansion was seen in the presence of IL-2 or IL-15.

Example 19 Inhibition of IL-28 and IL-29 Signaling with Soluble Receptor(zcytoR19/CRF2-4)

Signal Transduction Reporter Assay

A signal transduction reporter assay can be used to show the inhibitorproperties of zcytor19-Fc4 homodimeric and zcytor19-Fc/CRF2-4-Fcheterodimeric soluble receptors on zcyto20, zcyto21 and zcyto24signaling. Human embryonal kidney (HEK) cells overexpressing thezcytor19 receptor are transfected with a reporter plasmid containing aninterferon-stimulated response element (ISRE) driving transcription of aluciferase reporter gene. Luciferase activity following stimulation oftransfected cells with ligands (including zcyto20 (SEQ ID NO:2), zcyto21(SEQ ID NO:15), zcyto24 (SEQ ID NO:8)) reflects the interaction of theligand with soluble receptor.

Cell Transfections

293 HEK cells overexpressing zcytor19 were transfected as follows:700,000 293 cells/well (6 well plates) were plated approximately 18 hprior to transfection in 2 milliliters DMEM+10% fetal bovine serum. Perwell, 1 microgram pISRE-Luciferase DNA (Stratagene) and 1 microgrampIRES2-EGFP DNA (Clontech,) were added to 6 microliters Fugene 6 reagent(Roche Biochemicals) in a total of 100 microliters DMEM. Thistransfection mix was added 30 minutes later to the pre-plated 293 cells.Twenty-four hours later the transfected cells were removed from theplate using trypsin-EDTA and replated at approximately 25,000 cells/wellin 96 well microtiter plates. Approximately 18 h prior to ligandstimulation, media was changed to DMEM+0.5% FBS.

Signal Transduction Reporter Assays

The signal transduction reporter assays were done as follows: Followingan 18 h incubation at 37° C. in DMEM+0.5% FBS, transfected cells werestimulated with 10 ng/ml zcyto20, zcyto21 or zcyto24 and 10micrograms/ml of the following soluble receptors; human zcytor19-Fchomodimer, human zcytor19-Fc/human CRF2-4-Fc heterodimer, humanCRF2-4-Fc homodimer, murine zcytor19-Ig homodimer Following a 4-hourincubation at 37° C., the cells were lysed, and the relative light units(RLU) were measured on a luminometer after addition of a luciferasesubstrate. The results obtained are shown as the percent inhibition ofligand-induced signaling in the presence of soluble receptor relative tothe signaling in the presence of PBS alone. Table 16 shows that thehuman zcytor19-Fc/human CRF2-4 heterodimeric soluble receptor is able toinhibit zcyto20, zcyto21 and zcyto24-induced signaling between 16 and45% of control. The human zcytor19-Fc homodimeric soluble receptor isalso able to inhibit zcyto21-induced signaling by 45%. No significanteffects were seen with huCRF2-4-Fc or muzcytor19-Ig homodimeric solublereceptors.

TABLE 16 Percent Inhibition of Ligand-induced Interferon StimulatedResponse Element (ISRE) Signaling by Soluble Receptors Huzcytor19-Huzcytor19- Muzcytor19- Ligand Fc/huCRF2-4-Fc Fc HuCRF2-4-Fc Ig Zcyto2016% 92% 80% 91% Zcyto21 16% 45% 79% 103% Zcyto24 47% 90% 82% 89%

Example 20 Induction of Interferon Stimulated Genes by IL-28 and IL-29

Human Peripheral Blood Mononuclear Cells

Freshly isolated human peripheral blood mononuclear cells were grown inthe presence of IL-29 (20 ng/mL), IFNα2a (2 ng/ml) (PBL Biomedical Labs,Piscataway, N.J.), or in medium alone. Cells were incubated for 6, 24,48, or 72 hours, and then total RNA was isolated and treated withRNase-free DNase. 100 ng total RNA was used as a template for One-StepSemi-Quantitative RT-PCR® using Taqman One-Step RT-PCR Master Mix®Reagents and gene specific primers as suggested by the manufacturer.(Applied Biosystems, Branchburg, N.J.) Results were normalized to HPRTand are shown as the fold induction over the medium alone control foreach time-point. Table 17 shows that IL-29 induces Interferon StimulatedGene Expression in human peripheral blood mononuclear cells at alltime-points tested.

TABLE 17 MxA Fold Pkr Fold OAS Fold induction Induction Induction  6 hrIL29 3.1 2.1 2.5  6 hr IFNα2a 17.2 9.6 16.2 24 hr IL29 19.2 5.0 8.8 24hr IFNα2a 57.2 9.4 22.3 48 hr IL29 7.9 3.5 3.3 48 hr IFNα2a 18.1 5.017.3 72 hr IL29 9.4 3.7 9.6 72 hr IFNα2a 29.9 6.4 47.3

Activated Human T Cells

Human T cells were isolated by negative selection from freshly harvestedperipheral blood mononuclear cells using the Pan T-cell Isolation® kitaccording to manufacturer's instructions (Miltenyi, Auburn, Calif.). Tcells were then activated and expanded for 5 days with plate-boundanti-CD3, soluble anti-CD28 (0.5 ug/ml), (Pharmingen, San Diego, Calif.)and Interleukin 2 (IL-2; 100 U/ml) (R&D Systems, Minneapolis, Minn.),washed and then expanded for a further 5 days with IL-2. Followingactivation and expansion, cells were stimulated with IL-28A (20 ng/ml),IL-29 (20 ng/ml), or medium alone for 3, 6, or 18 hours. Total RNA wasisolated and treated with RNase-Free DNase. One-Step Semi-QuantitativeRT-PCR® was performed as described in the example above. Results werenormalized to HPRT and are shown as the fold induction over the mediumalone control for each time-point. Table 18 shows that IL-28 and IL-29induce Interferon Stimulated Gene expression in activated human T cellsat all time-points tested.

TABLE 18 MxA Fold Pkr Fold OAS Fold Induction Induction Induction Donor#1 3 hr IL28 5.2 2.8 4.8 Donor #1 3 hr IL29 5.0 3.5 6.0 Donor #1 6 hrIL28 5.5 2.2 3.0 Donor #1 6 hr IL29 6.4 2.2 3.7 Donor #1 18 hr IL28 4.64.8 4.0 Donor #1 18 hr IL29 5.0 3.8 4.1 Donor #2 3 hr IL28 5.7 2.2 3.5Donor #2 3 hr IL29 6.2 2.8 4.7 Donor #2 6 hr IL28 7.3 1.9 4.4 Donor #2 6hr IL29 8.7 2.6 4.9 Donor #2 18 hr IL28 4.7 2.3 3.6 Donor #2 18 hr IL294.9 2.1 3.8

Primary Human Hepatocytes

Freshly isolated human hepatocytes from two separate donors (Cambrex,Baltimore, Md. and CellzDirect, Tucson, Ariz.) were stimulated withIL-28A (50 ng/ml), IL-29 (50 ng/ml), IFNα2a (50 ng/ml), or medium alonefor 24 hours. Following stimulation, total RNA was isolated and treatedwith RNase-Free DNase. One-step semi-quantitative RT-PCR was performedas described previously in the example above. Results were normalized toHPRT and are shown as the fold induction over the medium alone controlfor each time-point. Table 19 shows that IL-28 and IL-29 induceInterferon Stimulated Gene expression in primary human hepatocytesfollowing 24-hour stimulation.

TABLE 19 MxA Fold Pkr Fold OAS Fold Induction Induction Induction Donor#1 IL28 31.4 6.4 30.4 Donor #1 IL29 31.8 5.2 27.8 Donor #1 IFN-α2a 63.48.2 66.7 Donor #2 IL28 41.7 4.2 24.3 Donor #2 IL29 44.8 5.2 25.2 Donor#2 IFN-α2a 53.2 4.8 38.3

HepG2 and HuH7: Human Liver Hepatoma Cell Lines

HepG2 and HuH7 cells (ATCC NOS. 8065, Manassas, Va.) were stimulatedwith IL-28A (10 ng/ml), IL-29 (10 ng/ml), IFNα2a (10 ng/ml), IFNB (1ng/ml) (PBL Biomedical, Piscataway, N.J.), or medium alone for 24 or 48hours. In a separate culture, HepG2 cells were stimulated as describedabove with 20 ng/ml of MetIL-29C172S-PEG or MetIL-29-PEG. Total RNA wasisolated and treated with RNase-Free DNase. 100 ng Total RNA was used asa template for one-step semi-quantitative RT-PCR as describedpreviously. Results were normalized to HPRT and are shown as the foldinduction over the medium alone control for each time-point. Table 20shows that IL-28 and IL-29 induce ISG expression in HepG2 and HuH7 liverhepatoma cell lines after 24 and 48 hours.

TABLE 20 MxA Fold Pkr Fold OAS Fold Induction Induction Induction HepG224 hr IL28 12.4 0.7 3.3 HepG2 24 hr IL29 36.6 2.2 6.4 HepG2 24 hr IFNα2a12.2 1.9 3.2 HepG2 24 hr IFNβ 93.6 3.9 19.0 HepG2 48 hr IL28 2.7 0.9 1.1HepG2 48 hr IL29 27.2 2.1 5.3 HepG2 48 hr IFNα2a 2.5 0.9 1.2 HepG2 48 hrIFNβ 15.9 1.8 3.3 HuH7 24 hr IL28 132.5 5.4 52.6 HuH7 24 hr IL29 220.27.0 116.6 HuH7 24 hr IFNα2a 157.0 5.7 67.0 HuH7 24 hr IFNβ 279.8 5.6151.8 HuH7 48 hr IL28 25.6 3.4 10.3 HuH7 48 hr IL29 143.5 7.4 60.3 HuH748 hr IFNα2a 91.3 5.8 32.3 HuH7 48 hr IFNβ 65.0 4.2 35.7

TABLE 21 MxA Fold OAS Fold Pkr Fold Induction Induction InductionMetIL-29-PEG 36.7 6.9 2.2 MetIL-29C172S-PEG 46.1 8.9 2.8

Data shown is for 20 ng/ml metlL-29-PEG and metIL-29C172S-PEG versionsof IL-29 after culture for 24 hours.

Data shown is normalized to HPRT and shown as fold induction overunstimulated cells.

Example 21 IL-28, IL-29, metIL-29-PEG and metIL-29C172S-PEG StimulateISG Induction in the Mouse Liver Cell Line AML-12

Interferon stimulated genes (ISGs) are genes that are induced by type Iinterferons (IFNs) and also by the IL-28 and IL-29 family molecules,suggesting that IFN and IL-28 and IL-29 induce similar pathways leadingto antiviral activity. Human type I IFNs (IFNα1-4 and IFNβ) have littleor no activity on mouse cells, which is thought to be caused by lack ofspecies cross-reactivity. To test if human IL-28 and IL-29 have effectson mouse cells, ISG induction by human IL-28 and IL-29 was evaluated byreal-time PCR on the mouse liver derived cell line AML-12.

AML-12 cells were plated in 6-well plates in complete DMEM media at aconcentration of 2×10⁶ cells/well. Twenty-four hours after platingcells, human IL-28 and IL-29 were added to the culture at aconcentration of 20 ng/ml. As a control, cells were either stimulatedwith mouse IFNα (positive control) or unstimulated (negative). Cellswere harvested at 8, 24, 48 and 72 hours after addition of CHO-derivedhuman IL-28A (SEQ ID NO:2) or IL-29 (SEQ ID NO:15). RNA was isolatedfrom cell pellets using RNAEasy-Kit® (Qiagen, Valencia, Calif.). RNA wastreated with DNase (Millipore, Billerica, Mass.) to clean RNA of anycontaminating DNA. cDNA was generated using Perkin-Elmer RT mix. ISGgene induction was evaluated by real-time PCR using primers and probesspecific for mouse OAS, Pkr and Mx1. To obtain quantitative data, HPRTreal-time PCR was duplexed with ISG PCR. A standard curve was obtainedusing known amounts of RNA from IFN-stimulated mouse PBLs. All data areshown as expression relative to internal HPRT expression.

Human IL-28A and IL-29 stimulated ISG induction in the mouse hepatocytecell line AML-12 and demonstrated that unlike type I IFNs, the IL-28/29family proteins showed cross-species reactivity.

TABLE 22 Stimulation OAS PkR Mx1 None 0.001 0.001 0.001 Human IL-28 0.040.02 0.06 Human IL-29 0.04 0.02 0.07 Mouse IL-28 0.04 0.02 0.08 MouseIFNα 0.02 0.02 0.01

All data shown were expressed as fold relative to HPRT gene expressionng of OAS mRNA=normalized value of OAS mRNA amount relative to internal

ng of HPRT mRNA housekeeping gene, HPRT

As an example, the data for the 48 hour time point is shown.

TABLE 23 AML12's Mx1 Fold OAS Fold Pkr Fold Induction InductionInduction MetIL-29-PEG 728 614 8 MetIL-29C172S-PEG 761 657 8

Cells were stimulated with 20 ng/ml metIL-29-PEG or metIL-29C172S-PEGfor 24 hours.

Data shown is normalized to HPRT and shown as fold induction overunstimulated cells.

Example 22 ISGs are Efficiently Induced in Spleens of Transgenic MiceExpressing Human IL-29

Transgenic (Tg) mice were generated expressing human IL-29 under thecontrol of the Eu-lck promoter. To study if human IL-29 has biologicalactivity in vivo in mice, expression of ISGs was analyzed by real-timePCR in the spleens of Eu-lck IL-29 transgenic mice.

Transgenic mice (C3H/C57BL/6) were generated using a construct thatexpressed the human IL-29 gene under the control of the Eu-lck promoter.This promoter is active in T cells and B cells. Transgenic mice andtheir non-transgenic littermates (n=2/gp) were sacrificed at about 10weeks of age. Spleens of mice were isolated. RNA was isolated from cellpellets using RNAEasy-Kit® (Qiagen). RNA was treated with DNase to cleanRNA of any contaminating DNA. cDNA was generated using Perkin-Elmer RT®mix. ISG gene induction was evaluated by real-time PCR using primers andprobes (5′ FAM, 3′ NFQ) specific for mouse OAS, Pkr and Mx1. To obtainquantitative data, HPRT real-time PCR was duplexed with ISG PCR.Furthermore, a standard curve was obtained using known amounts of IFNstimulated mouse PBLs. All data are shown as expression relative tointernal HPRT expression.

Spleens isolated from IL-29 Tg mice showed high induction of ISGs OAS,Pkr and Mx1 compared to their non-Tg littermate controls suggesting thathuman IL-29 is biologically active in vivo in mice.

TABLE 24 Mice OAS PkR Mx1 Non-Tg 4.5 4.5 3.5 IL-29 Tg 12 8 21

All data shown are fold expression relative to HPRT gene expression. Theaverage expression in two mice is shown

Example 23 Human IL-28 and IL-29 Protein Induce ISG Gene Expression InLiver, Spleen and Blood of Mice

To determine whether human IL-28 and IL-29 induce interferon stimulatedgenes in vivo, CHO-derived human IL-28A and IL-29 protein were injectedinto mice. In addition, E. coli derived IL-29 was also tested in in vivoassays as described above using MetIL-29C172S-PEG and MetIL-29-PEG. Atvarious time points and at different doses, ISG gene induction wasmeasured in the blood, spleen and livers of the mice.

C57BL/6 mice were injected i.p or i.v with a range of doses (10 μg-250μg) of CHO-derived human IL-28A and IL-29 or MetIL-29C172S-PEG andMetIL-29C16-C113-PEG. Mice were sacrificed at various time points (1hr-48 hr). Spleens and livers were isolated from mice, and RNA wasisolated. RNA was also isolated from the blood cells. The cells werepelleted and RNA isolated from pellets using RNAEasy®-kit (Qiagen). RNAwas treated with DNase (Amicon) to rid RNA of any contaminating DNA.cDNA was generated using Perkin-Elmer RT mix (Perkin-Elmer). ISG geneinduction was measured by real-time PCR using primers and probesspecific for mouse OAS, Pkr and Mx1. To obtain quantitative data, HPRTreal-time PCR was duplexed with ISG PCR. A standard curve was calculatedusing known amounts of IFN-stimulated mouse PBLs. All data are shown asexpression relative to internal HPRT expression.

Human IL-29 induced ISG gene expression (OAS, Pkr, Mx1) in the livers,spleen and blood of mice in a dose dependent manner. Expression of ISGspeaked between 1-6 hours after injection and showed sustained expressionabove control mice upto 48 hours. In this experiment, human IL-28A didnot induce ISG gene expression.

TABLE 25 Injection OAS-1 hr OAS-6 hr OAS-24 hr OAS-48 hr None - liver1.6 1.6 1.6 1.6 IL-29 liver 2.5 4 2.5 2.8 None - spleen 1.8 1.8 1.8 1.8IL-29 - spleen 4 6 3.2 3.2 None - blood 5 5 5 5 IL-29 blood 12 18 11 10

Results shown are fold expression relative to HPRT gene expression. Asample data set for IL-29 induced OAS in liver at a single injection of250 μg i.v. is shown. The data shown is the average expression from 5different animals/group.

TABLE 26 Injection OAS (24 hr) None 1.8 IL-29 10 μg 3.7 IL-29 50 μg 4.2IL-29 250 μg 6

TABLE 27 MetIL-29-PEG MetIL-29C172S-PEG Naive 3 hr 6 hr 12 hr 24 hr 3 hr6 hr 12 hr 24 hr 24 hr PKR 18.24 13.93 4.99 3.77 5.29 5.65 3.79 3.553.70 OAS 91.29 65.93 54.04 20.81 13.42 13.02 10.54 8.72 6.60 Mx1 537.51124.99 33.58 35.82 27.89 29.34 16.61 0.00 10.98

Mice were injected with 100 μg of proteins i.v. Data shown is foldexpression over HPRT expression from livers of mice. Similar data wasobtained from blood and spleens of mice.

Example 24 IL-28 and IL-29 Induce ISG Protein In Mice

To analyze of the effect of human IL-28 and IL-29 on induction of ISGprotein (OAS), serum and plasma from IL-28 and IL-29 treated mice weretested for OAS activity.

C57BL/6 mice were injected i.v with PBS or a range of concentrations (10μg-250 μg) of human IL-28 or IL-29. Serum and plasma were isolated frommice at varying time points, and OAS activity was measured using the OASradioimmunoassay (RIA) kit from Eiken Chemicals (Tokyo, Japan).

IL-28 and IL-29 induced OAS activity in the serum and plasma of miceshowing that these proteins are biologically active in vivo.

TABLE 28 Injection OAS-1 hr OAS-6 hr OAS-24 hr OAS-48 hr None 80 80 8080 IL-29 80 80 180 200

OAS activity is shown at pmol/dL of plasma for a single concentration(250 μg) of human IL-29.

Example 25 Signal Transduction Reporter Assay

A signal transduction reporter assay can be used to determine thefunctional interaction of human and mouse IL-28 and IL-29 with the IL-28receptor. Human embryonal kidney (HEK) cells are transfected with areporter plasmid containing an interferon-stimulated response element(ISRE) driving transcription of a luciferase reporter gene in thepresence or absence of pZP7 expression vectors containing cDNAs forclass II cytokine receptors (including human DIRS1, IFNαR1, IFNαR2 andIL-28 receptor). Luciferase activity following stimulation oftransfected cells with class II ligands (including IL-28A (SEQ ID NO:2), IL-29 (SEQ ID NO: 4), IL-28B (SEQ ID NO: 6), zcyto10, huIL10 andhuIFNa-2a) reflects the interaction of the ligand with transfected andnative cytokine receptors on the cell surface. The results and methodsare described below.

Cell Transfections

293 HEK cells were transfected as follows: 700,000 293 cells/well (6well plates) were plated approximately 18 h prior to transfection in 2milliliters DMEM+10% fetal bovine serum. Per well, 1 microgrampISRE-Luciferase DNA (Stratagene), 1 microgram cytokine receptor DNA and1 microgram pIRES2-EGFP DNA (Clontech,) were added to 9 microlitersFugene 6 reagent (Roche Biochemicals) in a total of 100 microlitersDMEM. Two micrograms pIRES2-EGFP DNA was used when cytokine receptor DNAwas not included. This transfection mix was added 30 minutes later tothe pre-plated 293 cells. Twenty-four hours later the transfected cellswere removed from the plate using trypsin-EDTA and replated atapproximately 25,000 cells/well in 96 well microtiter plates.Approximately 18 h prior to ligand stimulation, media was changed toDMEM+0.5% FBS.

Signal Transduction Reporter Assays

The signal transduction reporter assays were done as follows: Followingan 18 h incubation at 37° C. in DMEM+0.5% FBS, transfected cells werestimulated with dilutions (in DMEM+0.5% FBS) of the following class IIligands; IL-28A, IL-29, IL-28B, zcyto10, huIL10 and huIFNa-2a. Followinga 4-hour incubation at 37° C., the cells were lysed, and the relativelight units (RLU) were measured on a luminometer after addition of aluciferase substrate. The results obtained are shown as the foldinduction of the RLU of the experimental samples over the medium alonecontrol (RLU of experimental samples/RLU of medium alone=foldinduction). Table 29 shows that IL-28A, IL-29, and IL-28B induce ISREsignaling in 293 cells transfected with ISRE-luciferase giving a 15 to17-fold induction in luciferase activity over medium alone. The additionof IL-28 receptor alpha subunit DNA (SEQ ID NO:11), using the endogenousCRF2-4 (SEQ ID NO:71) to the transfection mix results in a 6 to 8-foldfurther induction in ISRE signaling by IL-28A, IL-29, and IL-28B givinga 104 to 125-fold total induction. None of the other transfected classII cytokine receptor DNAs resulted in increased ISRE signaling. Theseresults indicate that IL-28A, IL-29, and IL-28B functionally interactwith the IL-28 cytokine receptor. Table 29 also shows that huIFNa-2a caninduce ISRE signaling in ISRE-luciferase transfected 293 cells giving a205-fold induction of luciferase activity compared to medium alone.However, the addition of IL-28 receptor DNA to the transfection leads toan 11-fold reduction in ISRE-signaling (compared to ISRE-luciferase DNAalone), suggesting that IL-28 receptor over-expression negativelyeffects interferon signaling, in contrast to the positive effects ofIL-28 receptor over-expression on IL-28A, IL-29, and IL-28B signaling.

TABLE 29 Interferon Stimulated Response Element (ISRE) Signaling ofTransfected 293 Cells Following Class II Cytokine Stimulation (FoldInduction) Ligand ISRE-Luc. ISRE-Luc./IL-28R IL-28A (125 ng/ml) 15 125IL-29 (125 ng/ml) 17 108 IL-28B (125 ng/ml) 17 104 HuIFNa-2a (100 ng/ml)205 18 Zcyto10 (125 ng/ml) 1.3 1 HuIL10 (100 ng/ml) 1 0.5

Example 26 Signal Transduction Assays with IL-29 Cysteine Mutants

Cell Transfections

To produce 293 HEK cells stably overexpressing human IL-28 receptor, 293cells were transfected as follows: 300,000 293 cells/well (6 wellplates) were plated approximately 6 h prior to transfection in 2milliliters DMEM+10% fetal bovine serum. Per well, 2 micrograms of apZP7 expression vector containing the cDNA of human IL-28 receptor alphasubunit (SEQ ID NO: 11) was added to 6 microliters Fugene 6 reagent(Roche Biochemicals) in a total of 100 microliters DMEM. Thistransfection mix was added 30 minutes later to the pre-plated 293 cells.Forty-eight hours later the transfected cells were placed under 2microgram/milliliter puromicin selection. Puromicin resistant cells werecarried as a population of cells.

The 293 HEK cells overexpressing human IL-28 receptor were transfectedas follows: 700,000 293 cells/well (6 well plates) were platedapproximately 18 h prior to transfection in 2 milliliters DMEM+10% fetalbovine serum. Per well, 1 microgram KZ157 containing aninterferon-stimulated response element (ISRE) driving transcription of aluciferase reporter gene were added to 3 microliters Fugene 6 reagent(Roche Biochemicals) in a total of 100 microliters DMEM. Thistransfection mix was added 30 minutes later to the pre-plated 293HEKcells. Forty-eight hours later the transfected cells were removed fromthe plate using trypsin-EDTA and replated in 500 micrograms/ml G418(Geneticin, Life Technologies). Puromycin and G418 resistant cells werecarried as a population of cells.

Signal Transduction Reporter Assays

The signal transduction reporter assays were done as follows: 293HEKcells overexpressing human IL-28 receptor and containing KZ157 weretreated with trypsin-EDTA and replated at approximately 25,000cells/well in 96 well microtiter plates. Approximately 18 h prior toligand stimulation, media was changed to DMEM+0.5% FBS.

Following an 18 h incubation at 37° C. in DMEM+0.5% FBS, transfectedcells were stimulated with dilutions (in DMEM+0.5% FBS) of the differentforms of E. coli-derived zcyto21 containing different cysteine bindingpatterns. Following a 4-hour incubation at 37° C., the cells were lysed,and the relative light units (RLU) were measured on a luminometer afteraddition of a luciferase substrate. The results obtained are shown asthe fold induction of the RLU of the experimental samples over themedium alone control (RLU of experimental samples/RLU of mediumalone=fold induction).

Table 30 shows that C1-C3 form (C16-C113) of wild-type E. coli-derivedIL-29 is better able to induce ISRE signaling than wild-type C3-C5 form(C113-C172) or a mixture of wild-type C1-C3 form and C3-C5 form(C16-C113, C113-C172), all referring to SEQ ID NO:15.

Table 31 shows that C1-C3 (C16-C113) of wild-type E. coli-derived IL-29and C1-C3 (C16-C113; SEQ ID NO:15) of Cysteine mutant (C172S) E.coli-derived IL-29 (SEQ ID NO:29) are equally able to induce ISREsignaling in 293HEK cells overexpressing human IL-28 receptor.

TABLE 30 ISRE Signaling by different forms of E. coli-derived IL-29(Fold Induction) Cytokine Concentration C1-C3 form C3-C5 form Mixture ofC1-C3 (ng/ml) (C16-C113) (C113-C172) and C3-C5 100 36 29 34 10 38 25 351 32 12 24 0.1 10 2 5 0.01 3 1 1 0.001 1 1 1

TABLE 31 ISRE Signaling by different forms of E. coli-derived IL-29(Fold Induction) Cytokine Cysteine mutant Concentration Wild-type C172S(ng/ml) C1-C3 C1-C3 1000 9.9 8.9 100 9.3 8.7 10 9.3 8.1 1 7.8 7 0.1 4.63.3 0.01 1.9 1.5 0.001 1.3 0.9

Example 27 Human IL-29 Effect on B-Cells and IL-29 Toxic Saporin Fusion

The effects of human IL-29 are tested on the following human B-celllines: and human Burkitt's lymphoma cell lines Raji (ATCC No. CCL-86),and Ramos (ATCC No. CRL-1596); human EBV B-cell lymphoma cell line RPMI1788 (ATCC No. CRL-156); human myeloma/plasmacytoma cell line IM-9 (ATCCNo. CRL159); and human EBV transformed B-cell line DAKIKI (ATCC No.TIB-206), and HS Sultan cells (ATCC No. CRL-1484). Following about 2-5days treatment with IL-29, changes in surface marker expression on thecells shows that these cells can respond to IL-29. Human B-cell linestreated with IL-29 grow much more slowly than untreated cells whenreplated in cell culture dishes. These cells also have an increasedexpression of FAS ligand, as assessed by flow cytometry (Example 27D andExample 27E), and moderately increased sensitivity to an activating FASantibody (Example 27A). These results indicate that IL-29 could controlsome types of B-cell neoplasms by inducing them to differentiate to aless proliferative and or more FAS ligand sensitive state. Furthermore,IL-28 receptor is expressed on the surface of several B and T cell lines(Example 16). Thus, IL-29 and the human IL-29-saporin immunotoxinconjugate (Example 27B, below), or other IL-29-toxin fusion could betherapeutically used in B-cell leukemias and lymphomas.

The Effect of Human IL-29 on B-Cell Lines

IM-9 cells are seeded at about 50,000 cells per ml+/−50 μg/ml purifiedhuman IL-29. After 3 days growth the cells are harvested, washed andcounted then re-plated at about 2500 cells/ml in 96 well plates in towells with 0, 0.033, 0.1 or 0.33 μg/ml anti-FAS antibody (R&D Systems,Minneapolis). After 2 days an Alamar blue fluorescence assay isperformed (See U.S. Pat. No. 6,307,024) to assess proliferation of thecells.

The growth of IL-29 treated IM-9 cells is inhibited relative to thegrowth of untreated cells in the absence of anti-FAS antibody. In thepresence of 0.33 μg/ml anti-FAS antibody, the IL-29-treated cells areeven further inhibited.

The Effect of Human IL-29-Saporin Immunotoxin on B-Cell Lines

The human IL-29-saporin immunotoxin conjugate (IL-29-sap) constructionand purification is described in Example 28. The human IL-29-sap was farmore potent than the saporin alone in inhibiting cell growth. When thetreated cell are re-plated after a three or four day treatment the humanIL-29-sap treated cells grow very poorly.

IM-9, Ramos and K562 (ATCC No. CCL-243) cells are seeded at about 2500cells/well in 96 well plates with zero to 250 ng/ml human zalpha11L-sapconjugate or 0-250 ng/ml saporin (Stirpe et al., Biotechnology10:405-412, 1992) only as a control. The plates are incubated 4 daysthen an Alamar Blue proliferation assay is performed (U.S. Pat. No.6,307,024). At the maximal concentration of human IL-29-sap conjugate,the growth of cells is inhibited. Cells lines low/negative by flow forexpression of the IL-28 receptor are not affected by the IL-29-sap, thusshowing the specificity of the conjugate's effect.

IM-9 cells are seeded a 50,000 cells/ml into 6 well plates at zero and50 ng/ml human zalpha11L-sap conjugate. After 3 days the cells areharvested and counted then re-plated from 100 to 0.8 cells per well in 2fold serial dilutions, and 12 wells per cell dilution without the humanIL-29-saporin immunotoxin. After 6 days the number of wells with growthat each cell dilution is scored according to the results of an Alamarblue proliferation assay.

When cell number is assessed by Alamar blue assay the growth of thesurviving treated IM-9 cells is markedly impaired even after theremoval, by re-plating, of the IL-29-sap immunotoxin.

The limited tissue distribution of the human IL-28 receptor, and thespecificity of action of the IL-29-sap to receptor-expressing cell linessuggest that this conjugate may be tolerated in vivo.

The Effect of Human IL-29-Saporin Immunotoxin on B-Cell Line Viability

HS Sultan cells (ATCC No. CRL-1484) are seeded at about 40,000 cells perml into 12 well plates and grown for five days with either no addedcytokines or 40 ng/ml purified human IL-29 or 25 ng/ml human IL-29-sapconjugate (Example 28, below) or with 20 ng/ml IFN-alpha (RDI) or IL-29and IFN-alpha. IL-29 and IFN-alpha inhibit the outgrowth of the cellsindicating that the growth inhibitory effects of human IL-29 andIFN-alpha may be additive.

The results above support the possible use of IL-29 or human IL-29-sapin the treatment of malignancies or other diseases that express theIL-28 receptor, particularly those of B-cell origin. The combination ofIL-29 with IFN-alpha is specifically suggested by their additive effectin the inhibition of HS Sultan cells. Some other types of lymphoidmalignancies and diseases may also express the IL-28 receptor, asactivated T-cells also express the receptor mRNA and some of thesediseases may also be responsive to IL-29 of IL-29-toxic fusion therapy.

FAS (CD95) Expression on Human B-Cell Lines is Increased by Human IL-29Stimulation

Human B-cell lines HS Sultan (ATCC No. CRL-1484), IM-9 (ATCC No.CRL159), RPMI 8226 (ATCC No. CCL-155), RAMOS (ATCC No. CRL-1596), DAKIKI(ATCC No. TIB-206), and RPMI 1788 (ATCC No. CRL-156), are all treatedwith or without purified 10 to 50 ng/ml human IL-29 for 2 to 8 days. Thecells are then stained with anti-CD95 PE-conjugated antibody(PharMingen, San Diego, Calif.), per manufacturer's protocol, andanalyzed on a FACScalibur (Becton Dickinson, San Jose, Calif.). In allcell lines, anti-CD95 (FAS or APO-1) staining is increased upontreatment with human IL-29.

FAS (CD95) Expression on Primary Mouse Spleen B-Cells is Increased byHuman IL-29 Stimulation

Primary mouse splenocytes are obtained by chopping up spleens from 8 to12 week old C57/BL6 mice. Erythrocytes are lysed by treating thepreparation for 5 seconds with water then put through a 70 micron sieve.The remaining splenocytes are washed and plated in RPMI (JRH Bioscience)plus 10% HIA-FBS (Hyclone, Logan, Utah). IL-2 (R & D Systems) with orwithout human IL-29, as described above. They were then incubated at 37°C., in 5% CO₂ for 5 days. The splenocytes were harvested and stainedwith anti-CD95 PE conjugated antibody (PharMingen) and anti-CD19 FITCconjugated antibody (PharMingen) per manufacturer's protocol. The cellsare analyzed by flow cytometry on a FACScalibur (Becton Dickinson).

Example 28 Construction and Purification of IL-29 Toxic Fusion

Ten mg human IL-29 is conjugated to the plant toxin saporin (Stirpe etal., Biotechnology 10:405-412, 1992). The resulting 1.3 mg of a proteinconjugate is comprised of 1.1 molecules saporin per molecule of humanIL-29, formulated at a concentration of 1.14 mg/ml in 20 nM Sodiumphosphate, 300 nM sodium cloride, pH 7.2.

Example 29 IL-29 Toxic Fusion In Vivo

Testing IL-29-Saporin Conjugate in Mice

IL-29-saporin conjugate (Example 27) is administered to C57BL6 mice(female, 12 weeks of age, purchased from Taconic) at two differentdosages: 0.5 and 0.05 mg/kg. Injections are given i.v. in vehicleconsisting of 0.1% BSA (ICN, Costa Mesa, Calif.). Three injections aregiven over a period of one week (day 0, 2, and 7). Blood samples aretaken from the mice on day 0 (pre-injection) and on days 2 and 8(post-injection). Blood is collected into heparinized tubes (BectinDickenson, Franklin Lakes, N.J.), and cell counts are determined usingan automated hematology analyzer (Abbot Cell-Dyn model No. CD-3500CS,Abbot Park, Ill.). Animals are euthanized and necropsied on day 8following blood collection. Spleen, thymus, liver, kidney and bonemarrow are collected for histopathology. Spleen and thymus are weighed,and additional blood sample is collected in serum separator tubes. Serumis tested in a standard chemistry panel. Samples are also collected forflow cytometric analysis as described herein.

Testing IL-29 Toxic Saporin Fusion on B-Cell Derived Tumors In Vivo

The effects of human IL-29 and the human IL-29 toxic saporin fusion(Example 28) on human tumor cells are tested in vivo using a mouse tumorxenograft model described herein. The xenograft models are initiallytested using cell lines selected on the basis of in vitro experiments,such as those described in Example 27. These cell lines include, but arenot limited to: human Burkitt's lymphoma cell lines Raji (ATCC No.CCL-86), and Ramos (ATCC No. CRL-1596); human cell line RPMI 1788 (ATCCNo. CRL-156); human myeloma/plasmacytoma cell line IM-9 (ATCC No.CRL159); human cell line DAKIKI (ATCC No. TIB-206), and HS Sultan cells(ATCC No. CRL-1484). Cells derived directly from human tumors can alsobe used in this type of model. In this way, screening of patient samplesfor sensitivity to treatment with IL-29 or with a IL-29 toxic saporinfusion can be used to select optimal indications for use of zalpha11 inanti-cancer therapy.

After selection of the appropriate zenograft in vivo model, describedabove, IL-29-induced activity of natural killer cells and/or IL-29effects on B-cell derived tumors is assessed in vivo. Human IL-29 istested for its ability to generate cytotoxic effector cells (e.g., NKcells) with activity against B-cell derived tumors using mouse tumorxenograft models described herein. Moreover, direct affects of humanIL-29 on tumors can be assessed. The xenograft models to be carried outare selected as described above. A protocol using IL-29 stimulated humancells is developed and tested for efficacy in depleting tumor cells andpromoting survival in mice innoculated with cell lines or primarytumors.

Example 30 IL-29 Effect on B-Cell Derived Tumors In Vivo

Infusion of IL-29 Using Mini-Osmotic Pumps

Administration of IL-29 by constant infusion via mini-osmotic pumpsresults in steady state serum concentrations proportional to theconcentration of the IL-29 contained in the pump. 0.22 ml of human IL-29contained in phosphate buffered saline (pH 6.0) at a concentration of 2mg/ml or 0.2 mg/ml is loaded under sterile conditions into Alzetmini-osmotic pumps (model 2004; Alza corporation Palo Alto, Calif.).Pumps are implanted subcutaneously in mice through a 1 cm incision inthe dorsal skin, and the skin is closed with sterile wound closures.These pumps are designed to deliver their contents at a rate of 0.25 μlper hour over a period of 28 days. This method of administration resultsin significant increase in survival in mice injected with tumor cells(below).

IL-29 Effect on B-Cell Derived Tumors In Vivo

The effects of human IL-29 are tested in vivo using a mouse tumorxenograft model described herein. The xenograft model to be tested ishuman lymphoblastoid cell line IM-9 (ATCC No. CRL159). C.B-17 SCID mice(female C.B-17/IcrHsd-scid; Harlan, Indianapolis, Ind.) are divided into4 groups. On day 0, IM-9 cells (ATCC No. CRL159) are harvested fromculture and injected intravenously, via the tail vein, to all mice(about 1,000,000 cells per mouse). On day 1, mini-osmotic pumpscontaining test article or control article are implanted subcutaneouslyin the mice. Mice in groups 1-3 (n=9 per group) are treated withincreasing concentrations of IL-29: group 1 contains 2.0 mg/mL of humanIL-29 and is delivered 12 μg per day; group 2 contains 0.20 mg/mL ofhuman IL-29 and is delivered 1.2 μg per day; group 3 contained 0.02mg/mL of human IL-29 and is delivered 0.12 μg per day. Mice in group 4(n=9) are a control and are treated with vehicle (PBS pH 6.0).

Mice treated with either 12 μg/day or 1.2 μg/day IL-29 infusion haveincreased survival compared to vehicle treated mice (p<0.0001 andp<0.005 for 12 μg/day or 1.2 μg/day vs. vehicle, respectively, using logrank tests of the survival function). These results show that IL-29significantly reduced the effects of the B-cell tumor cells in vivo,significantly resulting in increased survival.

Example 31 In Vivo Anti-Tumor Effects of IL-29 in B16-F10 Melanoma andEG.7 Thymoma Models

Murine IL-29 Effect on B16-F10 Melanoma Metastasis Growth In Vivo

Mice (female, C57Bl6, 9 weeks old; Charles River Labs, Kingston, N.Y.)are divided into three groups. On day 0, B16-F10 melanoma cells (ATCCNo. CRL-6475) are harvested from culture and injected intravenously, viathe tail vein, to all mice (about 100,000 cells per mouse). Mice arethen treated with the test article or associated vehicle byintraperitoneal injection of 0.1 ml of the indicated solution. Mice inthe first group (n=24) are treated with vehicle (PBS pH 6.0), which isinjected on day 0, 2, 4, 6, and 8. Mice in the second group (n=24) aretreated with zcyto24 or zcyto25, which is injected at a dose of 75 μg onday 0, 2, 4, 6, and 8. Mice in the third group (n=12) are treated withzcyto24 or zcyto25, which is injected at a dose of 75 μg daily from day0 through day 9. All of the mice are sacrificed on day 18, and lungs arecollected for quantitation of tumor. Foci of tumor growth greater than0.5 mm in diameter are counted on all surfaces of each lung lobe. Inboth groups of mice treated with zcyto24 or zcyto25, the average numberof tumor foci present on lungs is significantly reduced, compared tomice treated with vehicle. Mice treated more frequently (i.e. daily)have fewer tumor foci than mice treated on alternate days.

These results indicated that treatment with zcyto24 or zcyto25 eitherslowed the growth of the B16 melanoma tumors or enhanced the ability ofthe immune system to destroy the tumor cells. The effects of thetreatment on tumor cells are likely mediated through cells of the immunesystem which do possess receptors for IL-29.

Murine IL-29 Effect on EG.7 Thymoma Growth In Vivo

Mice (female, C57Bl6, 9 weeks old; Charles River Labs, Kingston, N.Y.)are divided into three groups. On day 0, EG.7 cells (ATCC No. CRL-2113)are harvested from culture and 1,000,000 cells are injectedintraperitoneal in all mice. Mice are then treated with the test articleor associated vehicle by intraperitoneal injection of 0.1 mL of theindicated solution. Mice in the first group (n=6) are treated withvehicle (PBS pH 6.0), which is injected on day 0, 2, 4, and 6. Mice inthe second group (n=6) are treated with zcyto24 or zcyto25, which isinjected at a dose of 10 μg on day 0, 2, 4, and 6. Mice in the thirdgroup (n=6) are treated with zcyto24 or zcyto25, which is injected at adose of 75 mg on day 0, 2, 4, and 6. In both groups of mice treated withzcyto24 or zcyto25, time of survival is significantly increased,compared to mice treated with vehicle. These results indicate thattreatment with zcyto24 or zcyto25 either slowed the growth of the EG.7tumors or enhanced the ability of the immune system to destroy the tumorcells.

Example 32 Flow Cytometric Analysis IL-28 Receptor Expression

The expression of IL-28 receptors on neoplastic B cells derived fromnon-Hodgkin's lymphoma (NHL) specimens is assessed. Multiple MAbs areused to identify neoplastic B cells and to co-localize IL-28 receptors.The immunofluorescent staining by anti-IL-28 receptor MAb or bybiotin-IL-29 is recorded as mean peak fluorescence. The qualitativescores are assessed based on the shift in mean peak fluorescencerelative to an isotype matched control MAb.

Anti-IL-28 receptor MAb or biotin-IL-29 is used to detect IL-28 receptoron the neoplastic B cells by immunofluorescent staining. The intensityof the staining signal correlates to the levels of IL-28 receptor. Thesedata suggests that IL-28 receptors represent a therapeutic target fornon Hodgkin's lymphoma.

Example 33 In Vivo Effects of IL-29 on B-Cell Lymphomas

Human B-lymphoma cell lines are maintained in vitro by passage in growthmedium. The cells are washed thoroughly in PBS to remove culturecomponents.

SCID Mice are injected with (typically) one million human lymphoma cellsvia the tail vein in a 100 microliter volume. The optimal number of cellinjected is determined empirically in a pilot study to yield tumor takeconsistently with desired kinetics. IL-29 treatment is begun the nextday by either subcutaneous implantation of an ALZET® osmotic mini-pump(ALZET, Cupertino, Calif.) or by daily i.p. injection of IL-29 orvehicle. Mice are monitored for survival and significant morbidity. Micethat lose greater than 20% of their initial body weight are sacrificed,as well as mice that exhibit substantial morbidity such as hind limbparalysis. Depending on the lymphoma cell line employed, the untreatedmice typically die in 3 to 6 weeks. For B cell lymphomas that secreteIgG or IgM, the disease progression can also be monitored by weeklyblood sampling and measuring serum human Immunoglobulin levels by ELISA.

IL-29 Dose Response/IM-9 Model

Mice are injected with 1×10⁶ IM-9 cells, and 28 day osmotic mini pumpsimplanted the following day. The pumps are loaded with the followingconcentrations of IL-29 to deliver: 0, 0.12, 1.2 or 12 micrograms perday with 8 mice per dose group. IL-29 exhibits a clear dose dependenteffect in protecting mice from the tumor cell line. The effects of IL-29are dose dependent. Surviving mice at the end of the experiment have nosigns of disease and no detectable human IgG in their serum.

These data demonstrate that the efficacy of IL-29 in SCID mouse lymphomamodels correlates with the ability to inhibit the growth of the lymphomacell lines in vivo.

Example 34 The Effects of IL-29 in a Mouse Syngeneic Ovarian CarcinomaModel

The effect of IL-29 is tested for efficacy in ovarian carcinoma using amouse syngeneic model as described in Zhang et al., Am. J. of Pathol.161:2295-2309, 2002. Briefly, using retroviral transfection andfluorescence-activated cell sorting a C57BL6 murine ID8 ovariancarcinoma cell line is generated that stably overexpresses the murineVEGF164 isoform and the enhanced green fluorescence protein (GFP). Theretroviral construct containing VEGF164 and GFP cDNAs was transfectedinto BOSC23 cells. The cells are analyzed by FACS cell sorting and GFPhigh positive cells are identified.

The ID8 VEGF164/GFP transfected cells are cultured to subconfluence andprepared in a single-cell suspension in phosphate buffer saline (PBS)and cold MATRIGEL (BD Biosciences, Bedford, Mass.). Six to eight weekold femal C57BL6 mice are injected subcutaneously in the flank at 5×10⁶cells or untransfected control cells. Alternatively, the mice can beinjected intraperitoneally at 7×10⁶ cells or control cells. Animals areeither followed for survival or sacrificed eight weeks after inoculationand evaluated for tumor growth. Mice are treated with recombinantzcyto24 or zcyto25 beginning 3-14 days following tumor implantation, orwhen tumor engraftment and growth rate is established. Treatment levelsof 0.5-5 mg/kg will be administered on a daily basis for 5-14 days, andmay be continued thereafter if no evidence of neutralizing antibodyformation is seen.

Example 35 The Effects of IL-29 in a Mouse RENCA Model

The efficacy of IL-29 in a renal cell carcinoma model is evaluated usingBALB/c mice that have been injected with RENCA cells, a mouse renaladenocarcinoma of spontaneous origin, essentially as described inWigginton et al., J. Nat. Cancer Instit. 88:38-43, 1996.

Briefly, BALB/c mice between eight and ten weeks are injected with RENCAcells R 1×10⁵ cells into the kidney capsule of the mice. Twelve daysafter tumor cell implantation, the mice are nepharectomized to removeprimary tumors. The mice are allowed to recover from surgery, prior toadministration of IL-29. Mice are treated with recombinant zcyto24 orzcyto25 beginning 3-14 days following tumor implantation, or when tumorengraftment and growth rate is established. Treatment levels of 0.5-5mg/kg will be administered on a daily basis for 5-14 days, and may becontinued thereafter if no evidence of neutralizing antibody formationis seen. Alternatively, RENCA cells may be introduced by subcutaneous(5×10e5 cells) or intravenous (1×10e5 cells) injection.

The mice are evaluated for tumor response as compared to untreated mice.Survival is compared using a Kaplan-Meier method, as well as tumorvolume being evaluated.

Example 36 The Effects of IL-29 in a Mouse Colorectal Tumor Model

The effects of IL-29 in a colorectal mouse model are tested as describedin Yao et al., Cancer Res. 63:586-592, 2003. In this model, MC-26 mousecolon tumor cells are implanted into the splenic subcapsul of BALB/cmice. After 14 days, the treated mice are administered IL-29. Mice aretreated with recombinant zcyto24 or zcyto25 beginning 3-14 daysfollowing tumor implantation, or when tumor engraftment and growth rateis established. Treatment levels of 0.5-5 mg/kg will be administered ona daily basis for 5-14 days, and may be continued thereafter if noevidence of neutralizing antibody formation is seen.

The efficacy of IL-29 in prolonging survival or promoting a tumorresponse is evaluated using standard techniques described herein.

Example 37 The Effect of IL-29 in a Mouse Pancreatic Cancer Model

The efficacy of IL-29 in a mouse pancreatic cancer model is evaluatedusing the protocol developed by Mukherjee et al., J. Immunol.165:3451-3460, 2000. Briefly, MUC1 transgenic (MUC1.Tg) mice are bredwith oncogene-expressing mice that spontaneously develop tumors of thepancreas (ET mice) designated as MET. MUC1.Tg mice. ET mice express thefirst 127 aa of SV40 large T Ag under the control of the rat elastasepromoter. Fifty percent of the animals develop life-threateningpancreatic tumors by about 21 wk of age. Cells are routinely tested byflow cytometry for the presence of MUC1. All mice are on the C57BL/6background. Animals are sacrificed and characterized at 3-wk intervalsfrom 3 to 24 wk. Mice are carefully observed for signs of ill-health,including lethargy, abdominal distention, failure to eat or drink,marked weight loss, pale feces, and hunched posture.

The entire pancreas is dissected free of fat and lymph nodes, weighed,and spread on bibulus paper for photography. Nodules are counted, andthe pancreas is fixed in methacarn, processed for microscopy byconventional methods, step sectioned at 5 μm (about 10 sections permouse pancreas), stained with hematoxylin and eosin, and examined bylight microscopy. Tumors are obtained from MET mice at various timepoints during tumor progression, fixed in methacarn (60% methanol, 30%chloroform, 10% glacial acetic acid), embedded in paraffin, andsectioned for immunohistochemical analysis. MUC1 antibodies used areCT1, a rabbit polyclonal Ab that recognizes mouse and human cytoplasmictail region of MUC1, HMFG-2, BC2, and SM-3, which have epitopes in theTR domain of MUC1.

Determination of CTL activity is performed using a standard ⁵¹Cr releasemethod after a 6-day in vitro peptide stimulation without additionaladded cytokines. Splenocytes from individual MET mice are harvested bypassing through a nylon mesh followed by lysis of RBC.

Single cells from spleens of MET mice are analyzed by two-colorimmunofluorescence for alterations in lymphocyte subpopulations: CD3,CD4, CD8, Fas, FasL, CD11c, and MHC class I and II. Intracellularcytokine levels were determined after cells are stimulated with MUC1peptide (10 μg/ml for 6 days) and treated with brefeldin-A (also calledGolgi-Stop; PharMingen) as directed by the manufacturer's recommendation(4 μl/1.2×10⁷ cells/6 ml for 3 h at 37° C. before staining) Cells arepermeabilized using the PharMingen permeabilization kit and stained forintracellular IFN-γ, IL-2, IL-4, and IL-5 as described by PharMingen.All fluorescently labeled Abs are purchased from PharMingen. Flowcytometric analysis is done on Becton Dickinson FACscan using theCellQuest program (Becton Dickinson, Mountain View, Calif.).

Mice are treated with recombinant zcyto24 or zcyto25 beginning 3-14 daysfollowing tumor implantation, or when tumor engraftment and growth rateis established. Treatment levels of 0.5-5 mg/kg will be administered ona daily basis for 5-14 days, and may be continued thereafter if noevidence of neutralizing antibody formation is seen.

Example 38 The Effects of IL-29 in a Murine Breast Cancer Model

The efficacy of IL-29 in a murine model for breast cancer is made usinga syngeneic model as described in Colombo et al., Cancer Research62:941-946, 2002. Briefly, TS/A cells which are a spontaneous mammarycarcinoma for BALB/C mice. The cells are cultured for approximately oneweek to select for clones. The selected TS/A cells are grown and used tochallenge CD-1 nu/nu BR mice (Charles River Laboratories) by injected2×10² TS/A cells subcutaneously into the flank of the mouse.

Mice are treated with recombinant zcyto24 or zcyto25 beginning 3-14 daysfollowing tumor implantation, or when tumor engraftment and growth rateis established. Treatment levels of 0.5-5 mg/kg will be administered ona daily basis for 5-14 days, and may be continued thereafter if noevidence of neutralizing antibody formation is seen. The tumors areexcised after sacrificing the animals and analyzed for volume and usinghistochemistry and immunohistochemistry.

Example 39 The Effects of IL-29 in a Murine Prostate Cancer Model

The effects of IL-29 on tumor response are evaluated in murine prostatecancer model, using a model similar to that described in Kwon et al.,PNAS 96:15074-15079, 1999. In this model, there is a metastaticoutgrowth of transgenic adenocarcinoma of mouse prostate (TRAMP) derivedprostate cancer cell line TRAMP-C2, which are implanted in C57BL/6 mice.Metastatic relapse is reliable, occurring primarily in the draininglymph nodes in close proximity to the primary tumor.

Briefly, the C2 cell line used is an early passage line derived from theTRAMP mouse that spontaneously develops autochthonous tumorsattributable to prostate-restricted SV40 antigen expression. The cellsare cultured and injected subcutaneously into the C57BL/6 mice at2.5−5×10⁶ cells/0.1 ml media. Mice are treated with recombinant zcyto24or zcyto25 beginning 3-14 days following tumor implantation, or whentumor engraftment and growth rate is established. Treatment levels of0.5-5 mg/kg will be administered on a daily basis for 5-14 days, and maybe continued thereafter if no evidence of neutralizing antibodyformation is seen. The tumors are excised after sacrificing the animalsand analyzed for volume and using histochemistry andimmunohistochemistry.

Example 40 The Effects of IL-28 and IL-29 in the Murine ExperimentalAllergic Encephalomyelitis (EAE) Model

Experimental allergic encephalomyelitis (EAE) is a mouse model for humanMultiple Sclerosis (MS) (Gold et al., Mol. Med. Today, 6:88-91, 2000;Anderton et al., Immunol. Rev., 169:123-137, 1999). There are multipleways of inducing disease in mice. One such method is to immunize micewith a peptide of the myelin protein myelin oligodendrocyte glycoprotein(MOG). This protein is present on the outside of the myelin sheath andacts as a protective layer for myelin. Mice were immunizedsub-cutaneously with MOG peptide (MOG35-55) emulsified in RIBI adjuvanton day 0. Mice were then injected intravenously with pertussis toxin(PT) on day 2. The mice started showing symptoms of paralysis startingwith a limp tail, wobbly motion, followed by hind limb and forelimbparalysis, which were scored according to several different parametersthat measured the timing, extent and severity of disease. Delay in onsetof disease indicates that the drug is modifying the disease process inmice. Decrease in incidence indicates that the drug is having an effecton the number of mice that are getting sick. Decrease in clinical scoreindicates that the drug has an effect on the severity of disease. Groupsof mice were given PBS or either mouse IL28 (SEQ ID NO:8) or humanIL29C172S (SEQ ID NO:29)—PEG. The onset of symptoms, incidence ofdisease scores and severity of disease scores in IL-28/29 treated miceindicates the effect of IL-28/29 on these parameters in this model. Mice(n=13/gp) were immunized s.c with 100 ug MOG35-55 in RIBI adjuvant ond0. All mice received 200 ng pertussis toxin i.v on d2. Groups of micewere treated i.p with PBS, 25 ug human IL29C172S every other day (EOD)on days 1-18 or with PBS, BSA or mouse IL28. As specified above, micewere scored for clinical signs and weight loss daily from d0-d30. IL29C172S(SEQ ID NO:29)—PEG or mouse IL28 (SEQ ID NO:8) treated mice showeda delay in the onset of disease compared to PBS treated animals.

TABLE 32 P value Treatment groups Mean Day of (vs PBS group) D0-18 (EOD)Onset (MDO) Mantel-Cox test PBS 21.1 ± 4.7 — 25 ug human IL29 28.8 ± 4.50.0006 C172S-PEG

TABLE 33 Treatment groups P value (vs PBS group) Days 1-21 EOD Mean Dayof Onset (MDO) Mantel-Cox test PBS 8.6 ± 1.6 — 130 ug BSA 8.6 ± 1.3 NS130 ug mIL28 12.2 ± 3.3  P = 0.0009 (PBS) P = 0.001 (BSA) 

TABLE 34 Treatment groups P value (vs PBS group) Days 1-11 EOD Mean Dayof Onset (MDO) Mantel-Cox test PBS  9.5 ± 2.5 —  50 ug mIL28 12.4 ± 3.8P = 0.0354 200 ug mIL28 13.5 ± 3.2 P = 0.0007

IL-29 Delays Onset of Disease in a Mouse Model for Multiple Sclerosis

Summary

To test if human IL-29 had any effects on multiple sclerosis, theability of IL-29 to inhibit experimental autoimmune encephalomyelitis(EAE), a mouse model for MS was tested. The well characterized myelinoligodendrocyte glycoprotein (MOG) 35-55 peptide immunization model inC57BL/6 mice was used. The experiment was run to determine that IL-29could delay and/or inhibit disease scores in EAE. IL-29 delayed onset ofdisease in the EAE model, suggesting that use of IL-29 may be beneficialin MS.

Study Design

Experimental autoimmune encephalomyelitis (EAE) is a mouse model for MS.In one such model, C57BL/6 mice are immunized with 100 μg MOG peptide(MOG35-55) emulsified in RIBI adjuvant. Two milliliters of a 0.5 mg/mlpreparation of the MOG35-55 in PBS was added to a vial of RIBI andvortexed vigorously to emulsify the solution. The backs of mice wereshaved and 100 μg MOG/RIBI was injected s.c in the backs of mice.Weights of mice were taken 2 days before and every day after theimmunization. Mice were then injected on day 2 i.v with 200 μl pertussistoxin (PT), a final concentration of 200 ng/mouse. Mice were monitoreddaily for clinical scores. Groups of mice were injected i.p. with 200 μlPBS, or 25 ug IL-29 C172S (SEQ ID NO:29)—PEG in a 200 μl volume EOD fromdays 0-18. The weights of mice, clinical scores and incidence wereevaluated and plotted for analysis.

Results and Conclusion

Administration of IL-29 EOD from days 0-18 delayed onset of disease inthis model. This delay was significant compared to PBS treated mice(p=0.0006, Mantel-Cox test).

IL-28 Delays Onset of Disease in a Mouse Model for Multiple Sclerosis

Summary

To test if mouse IL-28 had any effects on multiple sclerosis, theability of IL-28 to inhibit experimental autoimmune encephalomyelitis(EAE), a mouse model for MS was tested. The well characterized myelinoligodendrocyte glycoprotein (MOG) 35-55 peptide immunization model inC57BL/6 mice was used. The experiment was run to determine that IL-28could delay and/or inhibit disease scores in EAE. IL-28 delayed onset ofdisease in the EAE model, suggesting that use of IL-28 may be beneficialin treatment of MS.

Study Design

Experimental autoimmune encephalomyelitis (EAE) is a mouse model for MS.In one such model, C57BL/6 mice are immunized with 100 μg MOG peptide(MOG35-55) emulsified in RIBI adjuvant. Two milliliters of a 0.5 mg/mlpreparation of the MOG35-55 in PBS was added to a vial of RIBI andvortexed vigorously to emulsify the solution. The backs of mice wereshaved and 100 μg MOG/RIBI was injected s.c in the backs of mice.Weights of mice were taken 2 days before and every day after theimmunization. Mice were then injected on day 2 i.v with 200 μl pertussistoxin (PT), a final concentration of 200 ng/mouse. Mice were monitoreddaily for clinical scores. In one experiment groups of mice wereinjected i.p. with 200 μl PBS, 50 ug mIL28 or 200 ug mIL28 (SEQ ID NO:8)in a 200 μl volume EOD from days 1-11. In a second experiment groups ofmice were injected i.p. with 200 μl PBS, 130 ug BSA or 130 ug mIL28 (SEQID NO:8) in a 200 μl volume EOD from days 1-21. The weights of mice,clinical scores and incidence were evaluated and plotted for analysis.

Results and Conclusion

Administration of IL-28 EOD delayed onset of disease in this model in adose dependent manner. This delay was significant compared to PBS or BSAtreated mice.

Example 41 IL-29 and IFNα2a MicroArray Comparison in Hepatoma Cell LineHepG2

Introduction

Type 1 interferons (IFNs) are induced following viral infection as partof the body's immune response to the virus. These proteins inhibit viralreplication through the induction of interferon-stimulated genes (ISGs)that act to directly inhibit viral replication, increase the lyticpotential of NK cells (Biron, C. A. 1998. Role of early cytokines,including alpha and beta interferons (IFN-alpha/beta), in innate andadaptive immune responses to viral infections. Semin Immunol 10:383-90)and modulate the adaptive immune response by increasing MHC class Iexpression to promote antigen presentation (Fellous, M., Nir, U.,Wallach, D., Merlin, G., Rubinstein, M., and Revel, M. 1982.Interferon-dependent induction of mRNA for the major histocompatibilityantigens in human fibroblasts and lymphoblastoid cells. Proc Natl AcadSci USA 79:3082-6), promoting T cell survival (Marrack, P., Kappler, J.,and Mitchell, T. 1999. Type I interferons keep activated T cells alive.J Exp Med 189:521-30) and stimulating dendritic cell maturation(Buelens, C., Bartholome, E. J., Amraoui, Z., Boutriaux, M., Salmon, I.,Thielemans, K., Willems, F., and Goldman, M. 2002. Interleukin-3 andinterferon beta cooperate to induce differentiation of monocytes intodendritic cells with potent helper T-cell stimulatory properties. Blood99:993-8). Because of this profound effect on the viral lifecycle,IFNα2a has proved to be a valuable therapeutic agent for the treatmentof Hepatitis C.

In addition to the type I interferons, viral infection induces theproduction of IL-28 and IL-29 (IFNλ 1-3), a recently discovered familyof novel class II cytokines distantly related to IFNα and IL-10. Likethe Type 1 IFNs IL28/29 have antiviral activity against a number ofviruses (Sheppard, P. et al., 2003. IL-28, IL-29 and their class IIcytokine receptor IL-28R. Nat Immunol 4:63-8; Kotenko, S. V. et al.,2003. IFN-lambdas mediate antiviral protection through a distinct classII cytokine receptor complex. Nat Immunol 4:69-77; and Robek, M. D. etal., 2005. Lambda interferon inhibits hepatitis B and C virusreplication. J Virol 79:3851-4). We and others have previously shownthat IL-29 induces the ISGs Mx1, PRKR and OAS in primary humanhepatocytes a well as human hepatoma cell lines such as HuH7 and HepG2.Therefore IL28/29 may regulate biology similar to IFNα2a and havetherapeutic value against chronic viral hepatitis in human patients.However, IL-29 and IFNα utilize distinct receptors making it possiblethat these two cytokines could potentially regulate othercytokine-specific genes subsets and biological processes. It wastherefore of interest to compare the gene regulation profiles of thesetwo cytokines on a global scale. Accordingly, HepG2 cells were treatedwith IL-29 and IFNα2a for varying times prior to isolation of total RNAand analysis of gene regulation using DNA microarray analysis.

Study Design

To identify genes regulated by IL-29 and IFNα2a in hepatocytes,microarray experiments were performed on the hepatoma cell line HepG2.For these studies triplicate cultures of HepG2 cells were treated withmedia as a negative control, 50 μg/ml human IL-29 (SEQ ID NO:4) or 5μg/ml human IFNα2a for one, six or twenty-four hours. Followingstimulation, total RNA was extracted using the RNeasy Mini kit fromQIAGEN and RNA quality and quantity were assessed on an Agilent 2100Bioanalyzer using the RNA 6000 Nano Assay (Agilent) according to themanufacturers instructions. Briefly, biotin-labeled cRNAs weresynthesized using the GeneChip® One-Cycle Target Labeling and ControlReagents from Affymetrix. Fragmented cRNA for each sample was hybridizedto Affymetrix Human Genome Focus Arrays and stained according to themanufacturer's instructions. Arrays were then scanned on an AffymetrixGeneChip® Scanner 3000 and raw data generated using Affymetrix GeneChip®Operating Software (GCOS) data mining software. Raw data was then loadedinto the GeneSpring 7.0 microarray analysis program (Silicon Genetics)for data analysis purposes. Values of less than 0.01 were transformed toa value of 0.01. The intensity of each array was normalized to the50^(th) percentile for all arrays using all values not absent and havinga raw value of 50 or greater. Values on a per gene basis were normalizedto the median calculated for values with a raw value of 50 or greater onall arrays. Scatter plots were generated using unfiltered data. Genesregulated by IL-29 were identified as having a 1-way analysis ofvariance (ANOVA) p-value of less than or equal to 0.05, a raw intensityin IL-29-treated samples of 600 (three times the background) or greaterand a fold change of 2 or greater as compared to the media-treatedsample at the corresponding time point. The most profound induction ofgenes was observed at the six-hour time point.

Results and Discussion

Upon analyzing the microarray results it was apparent that generegulation by both IL-29 and IFNα2a in HepG2 cells was transient,peaking at six hours followed by a gradual decline. After comparing thedata from the IL-29-treated sample to the data from the IFNα2a-treatedsample all genes were found to be regulated similarly by the twocytokines indicating that IL-29 and IFNα2a regulate identical genesubsets in hepatocytes. However, the degree of induction by IFNα2a inHepG2 cells was more profound than that elicited by IL-29. The list ofall genes identified as upregulated by IL-29 as determined by thecriteria listed in the Study Design is listed below in Table 35. Thesegenes were found to consist exclusively of known interferon-stimulatedgenes (ISGs) coding for proteins involved in antiviral responses (OASgenes, MX genes and PRKR, ADAR), regulation of proliferation (IFITM1,IFITM3, CEB1), apoptosis (TNFSF10) and signal transduction (NMI, STAT1,IRF9). These data suggest that IL-29 mediates biology identical to thatregulated by the type 1 interferons in cells such as hepatocytes thatexpress the IL-28 receptor.

TABLE 35 IFN Fold IL-29 Fold Gene Name Description Unigene ID ChangeChange IFIT1 interferon-induced protein with tetratricopeptide repeats 1Hs.20315 384.1 198.1 IFI27 interferon, alpha-inducible protein 27Hs.532634 221.5 91.96 OAS2 2′-5′ oligoadenylate synthetase 2 Hs.41433292.73 40.91 MX1 myxovirus (influenza virus) resistance 1 Hs.517307 81.4742.44 G1P3 interferon, alpha-inducible protein (clone IFI-6-16)Hs.523847 38.48 32.87 CEB1 cyclin-E binding protein 1 Hs.26663 34.094.526 IFIT3 interferon-induced protein with tetratricopeptide repeats 3Hs.47338 33.06 12.58 OAS1 2′,5′-oligoadenylate synthetase 1 Hs.52476026.78 13.1 OASL 2′-5′-oligoadenylate synthetase-like Hs.118633 25.878.516 OAS3 2′-5′-oligoadenylate synthetase 3 Hs.528634 23.15 10.83 MDA5melanoma differentiation associated protein-5 Hs.163173 22.7 7.423 G1P2interferon, alpha-inducible protein (clone IFI-15K) Hs.458485 22.49 13.6DDX58 DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 Hs.190622 21.63 8.265APOL6 apolipoprotein L, 6 Hs.257352 18.16 7.865 HSXIAPAF1 XIAPassociated factor-1 Hs.441975 15.2 7.96 NMI N-myc (and STAT) interactorHs.54483 13.85 3.855 PLSCR1 phospholipid scramblase 1 Hs.130759 11.646.899 UBE2L6 ubiquitin-conjugating enzyme E2L 6 Hs.425777 11.21 4.463SP110 SP110 nuclear body protein Hs.145150 10.94 4.551 USP18 ubiquitinspecific protease 18 Hs.38260 10.83 4.357 ISGF3G interferon regulatoryfactor 9 Hs.1706 10.44 7.496 STAT1 signal transducer and activator oftranscription 1, 91 kDa Hs.470943 9.701 5.565 SP100 Nuclear antigenSp100 Hs.369056 9.328 3.567 PSMB9 proteasome (prosome, macropain)subunit, beta type, 9 Hs.381081 9.227 3.128 TNFSF10 tumor necrosisfactor superfamily, member 10 (TRAIL) Hs.478275 8.819 3.003 MX2myxovirus (influenza virus) resistance 2 Hs.926 7.847 3.368 IFIT5interferon-induced protein with tetratricopeptide repeats 5 Hs.2528397.208 4.143 ISG20 interferon stimulated gene 20 kDa Hs.459265 7.1882.489 PRKR interferon-inducible double stranded RNA dependent proteinkinase Hs.131431 7.025 4.924 IFITM1 interferon induced transmembraneprotein 1 (9-27) Hs.458414 6.288 3.144 LY6E lymphocyte antigen 6complex, locus E (Sca-2) Hs.521903 4.047 2.282 BST2 bone marrow stromalcell antigen 2 Hs.118110 3.737 2.127 IFITM3 interferon inducedtransmembrane protein 3 (1-8U) Hs.374650 3.057 2.25

Example 42 Mouse IL28 Plasmid Inhibits Growth of Renal Cell CarcinomaRENCA Tumors in Mice

Summary

To determine whether IL28/IL29 has an effect on tumor growth in mice,groups of mice were injected s.c with the RENCA tumor on Day 0. Micewere then injected with 50 ug control vector plasmid or mIL28 plasmid(SEQ ID NO:7) by hydrodynamic delivery (HDD) on Days 5 and 12. Tumorvolume was monitored 3×/week for 5 weeks. Mouse IL28 protein level inserum was measured by ELISA. Mice injected with mIL28 plasmid showedsignificantly smaller tumors compared to control plasmid injected mice,suggesting that mouse IL28 has anti-tumor activity.

Study Design

Ten-week old female BALB/c mice (Charles River Laboratories) wereinjected s.c. on the right flank with 0.1×10⁶ RENCA cells on Day 0. Ondays 5 and 12, groups of mice (n=10/group) were injected i.v. with 50 ugof either empty pZP-7 plasmid or pZP-7/mL28 using the hydrodynamic pushmethod (inject plasmid resuspended in 1.6 ml of physiological saline viatail vein in 5-8 seconds). Mice were bled 24 hrs after plasmidinjections (Days 6 and 13) to assess serum mIL28 levels by ELISA. Tumorgrowth was monitored 3×/week for 5 weeks using caliper measurements.Tumor volume was calculated using the formula ½*(B)²*L (mm³)

Results and Conclusion

Injection of mIL28 plasmid resulted in protein expression between 50-200ng/ml 24 hours after plasmid delivery. Injection of mIL-28 plasmidinhibited tumor growth in the RENCA model. The differences in tumorvolume between control plasmid and IL28 plasmid injected mice wasstatistically significant (p=0.0125 compared to controls on Day 36)(FIG. 1). These data suggest that IL28 has anti-tumor activity and is apossible therapeutic for cancer.

Example 43 Mouse IL28 Plasmid and Human IL29 C172S-PEG Protein InhibitGrowth of RENCA Tumors in Mice

Summary

To determine if IL28/IL29 has an effect on tumor growth in mice, groupsof mice were injected s.c with the RENCA tumor on Day 0. Mice were theninjected with 50 ug control vector plasmid, mIL28 plasmid (SEQ ID NO:7)or mIFNα plasmid by hydrodynamic delivery (HDD) on Days 5 and 12. Aseparate group of tumor bearing mice received 25 ug human IL29 C172S(SEQ ID NO:29)—PEG (20 kD N-terminally conjugated methoxy-polyethyleneglycol propionaldehyde) protein by i.p. injection every other day (EOD)from Days 5-21. Tumor volume was monitored 3×/week for 4 weeks. MouseIL28 and IFNa protein levels in serum were measured by ELISA. Miceinjected with mIL28 or mIFNα plasmid showed significantly smaller tumorscompared to control plasmid injected mice, suggesting that mouse IL28has anti-tumor activity. Furthermore, mice injected with IL29 C172S-PEGprotein also showed decreased tumor volume compared to controls. Thesedata suggest that both IL28 and IL29 have anti-tumor activity.

Study Design

Ten-week old female BALB/c mice (Charles River Laboratories) wereinjected s.c. on the right flank with 0.1×10⁶ RENCA cells on Day 0. Ondays 5 and 12, groups of mice (n=10/group) were injected i.v. with 50 ugof either empty pZP-7 plasmid, pZP-7/mL28 or pORF/mIFNa using thehydrodynamic push method (inject plasmid resuspended in 1.6 ml ofphysiological saline via tail vein in 5-8 seconds). A separate group ofmice (n=10) were injected i.p. with 25 ug human IL29 C172S-PEG EOD fromdays 5-21. Intra-peritoneal injections were given in a total volume of200 ul. Mice were bled 24 hrs after plasmid injections (Days 6 and 13)to assess serum mIL28 and mIFNα levels by ELISA. Tumor growth wasmonitored 3×/week for 4 weeks using caliper measurements. Tumor volumewas calculated using the formula ½*(B)²*L (mm³)

Results and Conclusion

Administration of mIL-28 or mIFNα plasmid significantly inhibited tumorgrowth in this RENCA model (p<0.001 for all 3 groups compared to controlgroup on Day 28) (FIG. 2). Human IL-29 C172S-PEG protein injection alsosignificantly inhibited tumor growth compared to controls. These datasuggest that mIL28 and human IL29 have anti-tumor activity and arepossible therapeutics for cancer.

Example 44 Low Doses of 2 Different Forms of Human IL29 Protein ShowAnti-Tumor Activity in the RENCA Model

Summary

To determine if anti-tumor activity of IL29 can be achieved at lowerdoses than described above, groups of mice were injected s.c with theRENCA tumor on Day 0. Groups (n=10/group) of tumor bearing mice received1 ug, 5 ug, 25 ug human IL29 C172S (SEQ ID NO:29)—PEG (20 kDN-terminally conjugated methoxy-polyethylene glycol propionaldehyde) orhuman IL29 C172S d2-7 (SEQ ID NO:159)—PEG (20 kD N-terminally conjugatedmethoxy-polyethylene glycol propionaldehyde) protein by i.p. injectionevery other day (EOD) from Days 5-23. Tumor volume was monitored 3×/weekfor 4 weeks. Mice injected with 1, 5 or 25 ug IL29 C172S-PEG proteinshowed decreased tumor volume compared to controls. Furthermore, miceinjected with 1, 5 or 25 ug human IL29 C172S d2-7-PEG protein alsoshowed significantly decreased tumor growth compared to controls. Thesedata suggest that low doses of 2 different forms of human IL29 proteinhave anti-tumor activity in mice.

Study Design

Ten-week old female BALB/c mice (Charles River Laboratories) wereinjected s.c. on the right flank with 0.1×10⁶ RENCA cells on Day 0.Groups of mice (n=10/group) were injected i.p. with 1 ug, 5 ug, or 25 ughuman IL29 C172S-PEG or human IL29 C172S d2-7-PEG EOD from days 5-23.Intra-peritoneal injections were given in a total volume of 200 ul.Tumor growth was monitored 3×/week for 4 weeks using calipermeasurements. Tumor volume was calculated using the formula ½*(B)²*L(mm³)

Results and Conclusion

Administration of 1 ug, 5 ug or 25 ug human IL29 C172S-PEG proteinsignificantly inhibited tumor growth. Furthermore, 1 ug, 5 ug or 25 ugIL29 C172S d2-7-PEG protein injection also inhibited tumor growthcompared to vehicle treated mice (FIG. 3). These data provide evidencethat human IL29 protein has anti-tumor activity and is a potentialtherapeutic for various tumors.

Example 45 Therapeutic Treatment with Pegylated Human IL29 Shows PotentAnti-Tumor Activity in the RENCA Model

Summary

To determine if therapeutic treatment with IL29 can induce anti-tumoractivity groups of mice were injected s.c with the RENCA tumor on Day 0.When tumor volume of 100 mm³ was reached, mice received vehicle, 5 ug or25 ug human IL29 C172S d2-7 (SEQ ID NO:159)—PEG (20 kD N-terminallyconjugated methoxy-polyethylene glycol propionaldehyde) protein everyother day (EOD) for 10 injections or 5 ug human IL29 C172S d2-7 (SEQ IDNO:159)—PEG (20 kD N-terminally conjugated methoxy-polyethylene glycolpropionaldehyde) protein every day (ED) for 20 injections. As a control,one group of mice was treated prophylactically with 5 ug human IL29C172S d2-7-PEG EOD for 20 days starting on day 5 of tumor injection (Day5-23). Each individual mouse received injections only after its tumorvolume reached 100 mm³. All injections of protein were by i.p.administration. Tumor volume was monitored 3×/week for 4 weeks. Miceinjected with 5 ug or 25 ug EOD or 5 ug ED showed significantly lesstumor growth compared to controls. Consistent with previous results,mice given prophylactic treatment with 5 ug IL29 also showed decreasedtumor growth compared to controls. These data suggest that therapeutictreatment with human IL29 protein have anti-tumor activity in mice.

Study Design

Ten-week old female BALB/c mice (Charles River Laboratories) wereinjected s.c. on the right flank with 0.1×10⁶ RENCA cells on Day 0.Groups of mice (n=10/group) were injected i.p. with vehicle, 5 ug or 25ug human IL29 C172S d2-7-PEG EOD for 20 days or 5 ug human IL29 C172Sd2-7-PEG ED for 20 days starting with a tumor volume of approximately100 mm³. A separate group of mice received 5 ug human IL29 C172Sd2-7-PEG EOD for 20 days starting d5 of experiment (prophylactictreatment). Intra-peritoneal injections were given in a total volume of200 ul. Tumor growth was monitored 3×/week for 4 weeks using calipermeasurements. Tumor volume was calculated using the formula ½*(B)²*L(mm³)

Results and Conclusion

Mice injected with 5 ug or 25 ug EOD or 5 ug ED showed significantlyless tumor growth compared to controls. Consistent with previousresults, mice given prophylactic treatment with 5 ug IL29 also showeddecreased tumor growth compared to controls (FIG. 4). These data provideevidence that human IL29 protein has anti-tumor activity and is apotential therapeutic for various tumors.

Example 46 Prophylactic Treatment with Pegylated Human IL29 InhibitsTumor Growth in the E.G7 Thymoma Model

Summary

To determine if IL29 can induce anti-tumor activity in other tumors,groups of mice were injected s.c with the E.G7 tumor on Day 0. Groups ofmice received vehicle or 25 ug human IL29 C172S d2-7 (SEQ ID NO:159)—PEG(20 kD N-terminally conjugated methoxy-polyethylene glycolpropionaldehyde) protein every other day (EOD) for 10 injections (days0-18). All injections of protein were by i.p. administration. Tumorvolume was monitored 3×/week for 4 weeks. Mice injected with 25 ug EODshowed significantly less tumor growth compared to controls. These datasuggest that treatment with human IL29 protein have anti-tumor activityin mice.

Study Design

Ten-week old female C57BL/6 mice (Charles River Laboratories) wereinjected s.c. on the right flank with 0.4×10⁶ E.G7 cells on Day 0.Groups of mice (n=10/group) were injected i.p. with vehicle or 25 ughuman IL29 C172S d2-7-PEG EOD for 20 days. Intra-peritoneal injectionswere given in a total volume of 200 ul. Tumor growth was monitored3×/week for 4 weeks using caliper measurements. Tumor volume wascalculated using the formula ½*(B)²*L (mm³).

Results and Conclusion

Mice injected with 25 ug EOD showed significantly less tumor growthcompared to controls and also prolonged survival of mice compared tocontrol animals (FIGS. 5A and 5B). These data provide evidence thathuman IL29 protein has anti-tumor activity and is a potentialtherapeutic for various tumors.

Example 47 PEG-rIL-29, rIL-29, PEG-rIL-28A, rIL-28A, PEG-rIL-28B, orrIL-28B Inhibit Growth of Human Hepatocellular Carcinoma Cells C3A andHuH7 In Vivo

Summary

To test if anti-tumor activity of PEG-rIL-29, rIL-29, PEG-rIL-28A,rIL-28A, PEG-rIL-28B, or rIL-28B can be achieved against humanhepatocellular carcinoma cells in vivo, groups of BALB/c nude mice areinjected with either HuH7 or C3A hepatocellular carcinoma cells on Day0. Groups (n=10/group) of tumor bearing mice receive 5 ug-75 ug humanPEG-rIL-29, rIL-29, PEG-rIL-28A, rIL-28A, PEG-rIL-28B, or rIL-28Bprotein by i.p. or peritumoral injection every other day (EOD) from Days5-33. Tumor volume is monitored 3×/week for 6 weeks Inhibition of tumorgrowth by either PEG-rIL-29, rIL-29, PEG-rIL-28A, rIL-28A, PEG-rIL-28B,or rIL-28B protein suggests that the respective protein(s) hasinhibitory effects on human heptocellular carcinoma in vivo.

Study Design

Eight-week old female BALB/c nude mice (Charles River Laboratories) areinjected s.c. on the right flank with 6×10⁶ HuH7 or C3A cells on Day 0.Groups of mice (n=10/group) are injected i.p. or peritumorally with 5ug-75 ug human PEG-rIL-29, rIL-29, PEG-rIL-28A, rIL-28A, PEG-rIL-28B, orrIL-28B protein from days 5-33. Injections are given in a total volumeof 200 ul. Tumor growth is monitored 3×/week for 6 weeks using calipermeasurements. Tumor volume was calculated using the formula ½*(B)²*L(mm³)

Results and Conclusion

Inhibition of tumor growth by PEG-rIL-29, rIL-29, PEG-rIL-28A, rIL-28A,PEG-rIL-28B, or rIL-28B protein suggests that the respective protein(s)has inhibitory effects on human heptocellular carcinoma in vivo.

Example 48 PEG-rIL-29, rIL-29, PEG-rIL-28A, rIL-28A, PEG-rIL-28B, orrIL-28B Inhibit Growth of Human Prostate Carcinoma Cells PC-3 and DU145In Vivo

Summary

To test if anti-tumor activity of PEG-rIL-29, rIL-29, PEG-rIL-28A,rIL-28A, PEG-rIL-28B, or rIL-28B can be achieved against human prostatecarcinoma cells in vivo, groups of BALB/c nude mice are injected witheither PC-3 or DU-145 prostate carcinoma cells on Day 0. Groups(n=10/group) of tumor bearing mice receive 5 ug-75 ug human PEG-rIL-29,rIL-29, PEG-rIL-28A, rIL-28A, PEG-rIL-28B, or rIL-28B protein by i.p. orperitumoral injection every other day (EOD) from Days 5-33. Tumor volumeis monitored 3×/week for 6 weeks Inhibition of tumor growth (volume orweight) by PEG-rIL-29, rIL-29, PEG-rIL-28A, rIL-28A, PEG-rIL-28B, orrIL-28B protein suggests that respective protein has inhibitory effectson human prostate carcinoma in vivo.

Study Design

Eight-week old female BALB/c nude mice (Charles River Laboratories) areinjected s.c. on the right flank or orthotopically in the prostate lobewith 10×10⁶ PC-3 or 6×10⁶ DU-145 cells on Day 0. Groups of mice(n=10/group) are injected i.p. or peritumorally (s.c model only) with 5ug-75 ug human PEG-rIL-29, rIL-29, PEG-rIL-28A, rIL-28A, PEG-rIL-28B, orrIL-28B protein from days 5-33. Injections are given in a total volumeof 200 ul. For s.c tumors, tumor growth is monitored 3×/week for 6 weeksusing caliper measurements. Tumor volume was calculated using theformula ½*(B)²*L (mm³) For orthotopic tumors, mice are terminated at theend of the study and tumor weighed to enable tumor load assessment.

Results and Conclusion

Inhibition of tumor growth (volume or weight) by PEG-rIL-29, rIL-29,PEG-rIL-28A, rIL-28A, PEG-rIL-28B, or rIL-28B protein suggests that therespective protein(s) has inhibitory effects on human prostate carcinomain vivo.

Example 49 PEG-rIL-29, rIL-29, PEG-rIL-28A, rIL-28A, PEG-rIL-28B, orrIL-28B Inhibit Growth of Human Colon Carcinoma Cells DLD-1 and HCT-116In Vivo

Summary

To test if anti-tumor activity of PEG-rIL-29, rIL-29, PEG-rIL-28A,rIL-28A, PEG-rIL-28B, or rIL-28B can be achieved against human coloncarcinoma cells in vivo, groups of BALB/c nude mice are injected witheither DLD-1 or HCT-116 colon carcinoma cells on Day 0. Groups(n=10/group) of tumor bearing mice receive 5 ug-75 ug human PEG-rIL-29,rIL-29, PEG-rIL-28A, rIL-28A, PEG-rIL-28B, or rIL-28B protein by i.p. orperitumoral injection every other day (EOD) from Days 5-33. Tumor volumeis monitored 3×/week for 6 weeks. Inhibition of tumor growth (volume orweight) by PEG-rIL-29, rIL-29, PEG-rIL-28A, rIL-28A, PEG-rIL-28B, orrIL-28B protein suggests that the respective protein has inhibitoryeffects on human colon carcinoma in vivo.

Study Design

Eight-week old female BALB/c nude mice (Charles River Laboratories) areinjected s.c. on the right flank or orthotopically in the colonic wallwith 6×10⁶ DLD-1 or HCT-116 cells on Day 0. Groups of mice (n=10/group)are injected i.p. or peritumorally (for s.c model only) with 5 ug-75 ughuman PEG-rIL-29, rIL-29, PEG-rIL-28A, rIL-28A, PEG-rIL-28B, or rIL-28Bprotein from days 5-33. Injections are given in a total volume of 200ul. For s.c tumors, tumor growth is monitored 3×/week for 6 weeks usingcaliper measurements. Tumor volume was calculated using the formula½*(B)²*L (mm³) For orthotopic tumors, mice are terminated at the end ofthe study and tumor weighed to enable tumor load assessment.

Results and Conclusion

Inhibition of tumor growth (volume or weight) by PEG-rIL-29, rIL-29,PEG-rIL-28A, rIL-28A, PEG-rIL-28B, or rIL-28B protein suggests that therespective protein(s) has inhibitory effects on human colon carcinoma invivo.

Example 50 PEG-rIL-29, rIL-29, PEG-rIL-28A, rIL-28A, PEG-rIL-28B, orrIL-28B Inhibit Growth of Human Pancreatic Carcinoma Cells BxPC-3 andHPAF-II In Vivo

Summary

To test if anti-tumor activity of PEG-rIL-29, rIL-29, PEG-rIL-28A,rIL-28A, PEG-rIL-28B, or rIL-28B can be achieved against humanpancreatic carcinoma cells in vivo, groups of BALB/c nude mice areinjected with either BxPC-3 or HPAF-II pancreatic carcinoma cells on Day0. Groups (n=10/group) of tumor bearing mice receive 5 ug-75 ug humanPEG-rIL-29, rIL-29, PEG-rIL-28A, rIL-28A, PEG-rIL-28B, or rIL-28Bprotein by i.p. or peritumoral injection every other day (EOD) from Days5-33. Tumor volume is monitored 3×/week for 6 weeks Inhibition of tumorgrowth (volume or weight) by PEG-rIL-29, rIL-29, PEG-rIL-28A, rIL-28A,PEG-rIL-28B, or rIL-28B protein suggests that the respective protein hasinhibitory effects on human pancreatic carcinoma in vivo.

Study Design

Eight-week old female BALB/c nude mice (Charles River Laboratories) areinjected s.c. on the right flank or orthotopically in the pancreaticlobe with 6×10⁶ BxPC-3 or HCT-116 cells on Day 0. Groups of mice(n=10/group) are injected i.p. or peritumorally (for s.c model only)with 5 ug-75 ug human PEG-rIL-29, rIL-29, PEG-rIL-28A, rIL-28A,PEG-rIL-28B, or rIL-28B protein from days 5-33. Injections are given ina total volume of 200 ul. For s.c tumors, tumor growth is monitored3×/week for 6 weeks using caliper measurements. Tumor volume wascalculated using the formula ½*(B)²*L (mm³) For orthotopic tumors, miceare terminated at the end of the study and tumor weighed to enable tumorload assessment.

Results and Conclusion

Inhibition of tumor growth (volume or weight) by PEG-rIL-29, rIL-29,PEG-rIL-28A, rIL-28A, PEG-rIL-28B, or rIL-28B protein suggests that therespective protein(s) has inhibitory effects on human pancreaticcarcinoma in vivo.

Example 51 IL-29 in EAE Mouse Models for Multiple Sclerosis (MS)

Experimental allergic encephalomyelitis (EAE) is a mouse model for humanMS (Gold et al., Mol. Med. Today, 6:88-91, 2000; Anderton et al.,Immunol. Rev., 169:123-137, 1999). MS in humans can be broadlyclassified into chronic progressive and relapsing remitting diseasephenotypes. These disease phenotypes can be modeled in mice usingmultiple methods. One such method of inducing chronic progressive MS inmice is to immunize mice with a peptide of the myelin protein MOG(myelin oligodendrocyte glycoprotein). This protein is present on theoutside of the myelin sheath and acts as a protective layer for myelin.Mice are immunized sub-cutaneously with MOG peptide (MOG35-55)emulsified in RIBI adjuvant on day 0. Mice are then injectedintravenously with pertussis toxin (PT) on day 2. The mice start showingsymptoms of paralysis starting with a limp tail, wobbly motion, followedby hind limb and forelimb paralysis, which are scored according toseveral different parameters that measure the timing, extent andseverity of disease. Delay in onset of disease indicates that the drugis modifying the disease process in mice. Decrease in incidenceindicates that the drug is having an effect on the number of mice thatare getting sick. Decrease in clinical score indicates that the drug hasan effect on the severity of disease. Groups of mice are given PBS oreither mouse IL28 or human IL29C172S-PEG. The onset of symptoms,incidence of disease scores and severity of disease scores in IL-28/29treated mice indicates the effect of IL-28/29 on these parameters inthis model. Mice (n=13/gp) are immunized s.c with 100 ug MOG35-55 inRIBI adjuvant on d0. All mice receive 200 ng pertussis toxin i.v on d2.Groups of mice are treated i.p with PBS, 25 ug human IL29C172S everyother day (EOD) on days 1-18 or with PBS, BSA or mouse IL28. Asspecified above, mice are scored for clinical signs and weight lossdaily from d0-d30. IL29 C172S-PEG or mouse IL28 treated mice show adelay in the onset of disease compared to PBS treated animals.

To model relapsing remitting disease, mice (SJL) are immunized with apeptide derived from the proteolipid protein (PLP). Mice are immunizedin the back s.c with PLP139-151 peptide emulsified in complete Freund'sadjuvant (CFA). The mice start showing symptoms of paralysis startingwith a limp tail, wobbly motion, followed by hind limb and forelimbparalysis, which are scored according to several different parametersthat measure the timing, extent and severity of disease. In the RR-EAEmodel, during the course of disease, mice will spontaneously have aremission for a short period after which mice will again relapsespontaneously. Mice might have these relapsing-remitting cycles multipletimes during the course of the disease. Delay in onset of diseaseindicates that the drug is modifying the disease process in mice.Decrease in incidence indicates that the drug is having an effect on thenumber of mice that are getting sick. Decrease in clinical scoreindicates that the drug has an effect on the severity of disease. Inthis model, decreases in numbers of relapses, decrease in maximumclinical score achieved at a relapse and to maintain mice in completeremission are all indications of a therapeutic drug. Groups of immunizedmice are given either prophylactic (starting Day 3 after immunization)or therapeutic (starting on first day of clinical score) PEG-rIL-29 orPEG-mIL-28 at different doses. Decrease in relapses, disease severityand incidence or induction of complete remission indicate thatPEG-rIL-29 or PEG-mIL-28 can inhibit RR-EAE and could be a therapeuticfor RR-MS in humans.

Prophylactic Administration of PEG-mIL-28 or PEG-rIL-29 InhibitsSeverity of Disease and Disease Incidence in the RR-EAE Model in SJLMice

Summary

To test if mouse IL-28 or human IL-29 had any effects onrelapsing-remitting multiple sclerosis, the ability of PEG-mIL-28 orPEG-rIL-29 to inhibit experimental autoimmune encephalomyelitis (EAE), amouse model for RR-MS was tested. The well characterized proteolipidprotein PLP 139-151 peptide immunization model in SJL mice was used. Theexperiment was run to determine that IL-28 or IL-29 could delay and/orinhibit disease scores in RR-EAE. Prophylactic administration of eitherPEG-rIL-29 and PEG-mIL-28 delayed onset of disease and reduced incidenceof disease in the RR-EAE model, suggesting that use of IL-28 or IL-29may be beneficial in MS.

Study Design

Experimental autoimmune encephalomyelitis (EAE) is a mouse model for MS.In one such model, SJL mice are immunized with 100 μg PLP peptide(PLP139-151) emulsified in CFA adjuvant (1.1 ratio). A 1 mg/mLpreparation of the PLP139-151 peptide in PBS was prepared. CFA (SigmaAldrich Ltd) containing 1 mg/mL heat inactivated mycobacteriumtuberculosis (Mtb) was fortified with further 1 mg/mL of Mtb (DifcoLaboratories) to make a final concentration of 2 mg/mL Mtb. A 1:1emulsion of this CFA and PLP peptide solution was generated usingemulsifying syringes. The backs of mice were shaved and 100 μg PLP/CFA(200 uL of emulsion) was injected s.c in the backs of mice. Weights ofmice were taken 2 days before and every day after the immunization. Micewere monitored daily for clinical scores. Groups of mice were injectedi.p. with 100 μl PBS, or 5-75 ug PEG-mIL-28 (mouse IL-28 (SEQ IDNO:8)—PEG (20 kD N-terminally conjugated methoxy-polyethylene glycolpropionaldehyde) or 25 ug PEG-rIL-29 (IL29 C172S d2-7 (SEQ IDNO:159)—PEG (20 kD N-terminally conjugated methoxy-polyethylene glycolpropionaldehyde) in a 100 uL volume EOD from days 3-23. In someexperiments, 25 ug Novantrone was administered i.p. on Days 4, 8, 12 and16. The weights of mice, clinical scores and incidence were evaluatedand plotted for analysis.

Results and Conclusion

Prophylactic administration of 5 ug, 25 ug or 75 ug PEG-mIL-28significantly inhibited disease incidence, and delayed disease onset inthe RR-EAE model (See FIGS. 6 and 7, p<0.0001 for all PEG-mIL-28 groupsvs Vehicle—Kaplein-Meier test). Prophylactic administration of 25 ugPEG-rIL-29 significantly inhibited disease incidence and delayed diseaseonset in the RR-EAE model (FIGS. 8 and 9, p<0.0001 compared to vehicle,Kaplein Meier test)

Therapeutic Administration of PEG-mIL-28 Inhibits Severity of Diseaseand Relapse of Disease in the RR-EAE Model in SJL Mice

Summary

To test if therapeutic administration of mouse IL-28 had any effects onrelapsing-remitting multiple sclerosis, the ability of PEG-mIL-28 toinhibit experimental autoimmune encephalomyelitis (EAE), a mouse modelfor RR-MS was tested. The well characterized proteolipid protein PLP139-151 peptide immunization model in SJL mice was used. The experimentwas run to determine that IL-28 could inhibit disease scores and preventrelapses when given therapeutically (after first clinical sign ofdisease) in RR-EAE. Therapeutic administration of PEG-mIL-28 inhibiteddisease severity and reduced relapses in the RR-EAE model, suggestingthat use of IL-28 may be beneficial in MS.

Study Design

Experimental autoimmune encephalomyelitis (EAE) is a mouse model for MS.In one such model, SJL mice were immunized with 100 μg PLP peptide(PLP139-151) emulsified in CFA adjuvant (1.1 ratio). A 1 mg/mLpreparation of the PLP139-151 peptide in PBS was prepared. CFA (SigmaAldrich Ltd) containing 1 mg/mL heat inactivated mycobacteriumtuberculosis (Mtb) was fortified with further 1 mg/mL of Mtb (DifcoLaboratories) to make a final concentration of 2 mg/mL Mtb. A 1:1emulsion of this CFA and PLP peptide solution was generated usingemulsifying syringes. The backs of mice were shaved and 100 μg PLP/CFA(200 uL of emulsion) was injected s.c in the backs of mice. Weights ofmice were taken 2 days before and every day after the immunization. Micewere monitored daily for clinical scores. Groups of mice were injectedi.p. with 100 μl PBS, or 25 ug or 50 ug PEG-mIL-28 (mouse IL-28 (SEQ IDNO:8)—PEG (20 kD N-terminally conjugated methoxy-polyethylene glycolpropionaldehyde) in a 100 uL volume every day (ED) for 30 days startingfrom Day 1 of first clinical score in each individual mice. The weightsof mice, clinical scores and incidence were evaluated and plotted foranalysis.

Results and Conclusion

Therapeutic administration of 25 ug or 50 ug PEG-mIL-28 significantlyinhibited disease severity, in the RR-EAE model (FIGS. 10 and 11).PEG-mIL-28 also reduced relapses and sustained remission in RR-EAE mice(FIG. 12)

Therapeutic Administration of PEG-rIL-29 Inhibits Severity of Diseaseand Relapse of Disease in the RR-EAE Model in SJL Mice

Summary

To test if therapeutic administration of human IL-29 had any effects onrelapsing-remitting multiple sclerosis, the ability of PEG-rIL-29 toinhibit experimental autoimmune encephalomyelitis (EAE), a mouse modelfor RR-MS was tested. The well characterized proteolipid protein PLP139-151 peptide immunization model in SJL mice was used. The experimentwas run to determine that IL-29 could inhibit disease scores and preventrelapses when given therapeutically (after first clinical sign ofdisease) in RR-EAE. Inhibition of disease severity and prevention ofrelapses in RR-EAE model by PEG-rIL-29 suggest the clinical use of thisprotein in human MS.

Study Design

Experimental autoimmune encephalomyelitis (EAE) is a mouse model for MS.In one such model, SJL mice are immunized with 100 μg PLP peptide(PLP139-151) emulsified in CFA adjuvant (1.1 ratio). A 1 mg/mLpreparation of the PLP139-151 peptide in PBS was prepared. CFA (SigmaAldrich Ltd) containing 1 mg/mL heat inactivated mycobacteriumtuberculosis (Mtb) was fortified with further 1 mg/mL of Mtb (DifcoLaboratories) to make a final concentration of 2 mg/mL Mtb. A 1:1emulsion of this CFA and PLP peptide solution was generated usingemulsifying syringes. The backs of mice were shaved and 100 μg PLP/CFA(200 uL of emulsion) was injected s.c in the backs of mice. Weights ofmice were taken 2 days before and every day after the immunization. Micewere monitored daily for clinical scores. Groups of mice were injectedi.p. with 100 μl PBS, or 5 ug-75 ug PEG-rIL-29 (IL29 C172S d2-7 (SEQ IDNO:159)—PEG (20 kD N-terminally conjugated methoxy-polyethylene glycolpropionaldehyde) in a 100 uL volume every day (ED) or every other day(EOD) for 30 days starting from Day 1 of first clinical score in eachindividual mice. The weights of mice, clinical scores and incidence wereevaluated and plotted for analysis.

Results and Conclusion

After therapeutic administration of PEG-rIL-29, the disease severity andprevention of relapses in RR-EAE model was inhibited. This suggestsclinical use of this protein in human MS.

The complete disclosure of all patents, patent applications, andpublications, and electronically available material (e.g., GenBank aminoacid and nucleotide sequence submissions) cited herein are incorporatedby reference. The foregoing detailed description and examples have beengiven for clarity of understanding only. No unnecessary limitations areto be understood therefrom. The invention is not limited to the exactdetails shown and described, for variations obvious to one skilled inthe art will be included within the invention defined by the claims.

1. A method of inhibiting the disease severity of relapsing-remittingmultiple sclerosis in a mammal comprising administering to the mammal atherapeutically effective amount of a polypeptide encoded by apolynucleotide of SEQ ID NO:174, wherein the encoded polypeptidecomprises amino acid residues 1-176 of SEQ ID NO:175.
 2. The method ofclaim 1 wherein the polypeptide is conjugated to a polyalkyl oxidemoiety.
 3. The method of claim 2 wherein the polyalkyl oxide moiety ispolyethylene glycol.
 4. The method of claim 3 wherein the polyethyleneglycol is monomethoxy-PEG propionaldehyde.
 5. The method of claim 4wherein the monomethoxy-PEG propionaldehyde has a molecular weight ofabout 20 Kd or 30 Kd.
 6. The method of claim 4 wherein themonomethoxy-PEG propionaldehyde is linear or branched.
 7. The method ofclaim 4 wherein the monomethoxy-PEG propionaldehyde is conjugatedN-terminally to the polypeptide.
 8. A method of inhibiting the diseaseseverity of relapsing-remitting multiple sclerosis in a mammalcomprising administering to the mammal a therapeutically effectiveamount of a formulation comprising a polypeptide encoded by apolynucleotide of SEQ ID NO:174 and a pharmaceutically acceptablevehicle, wherein the encoded polypeptide comprises amino acid residues1-176 of SEQ ID NO:175.
 9. The method of claim 8 wherein the polypeptideis conjugated to a polyalkyl oxide moiety.
 10. The method of claim 9wherein the polyalkyl oxide moiety is polyethylene glycol.
 11. Themethod of claim 10 wherein the polyethylene glycol is monomethoxy-PEGpropionaldehyde.
 12. The method of claim 11 wherein the monomethoxy-PEGpropionaldehyde has a molecular weight of about 20 Kd or 30 Kd.
 13. Themethod of claim 11 wherein the monomethoxy-PEG propionaldehyde is linearor branched.
 14. The method of claim 11 wherein the monomethoxy-PEGpropionaldehyde is conjugated N-terminally to the polypeptide.
 15. Amethod of delaying the onset of relapsing-remitting multiple sclerosisin a mammal comprising administering to the mammal a therapeuticallyeffective amount of a polypeptide encoded by a polynucleotide of SEQ IDNO:174, wherein the encoded polypeptide comprises amino acid residues1-176 of SEQ ID NO:175.
 16. The method of claim 15 wherein thepolypeptide is conjugated to a polyalkyl oxide moiety.
 17. The method ofclaim 16 wherein the polyalkyl oxide moiety is polyethylene glycol. 18.The method of claim 17 wherein the polyethylene glycol ismonomethoxy-PEG propionaldehyde.
 19. The method of claim 18 wherein themonomethoxy-PEG propionaldehyde has a molecular weight of about 20 Kd or30 Kd.
 20. The method of claim 18 wherein the monomethoxy-PEGpropionaldehyde is linear or branched.
 21. The method of claim 18wherein the monomethoxy-PEG propionaldehyde is conjugated N-terminallyto the polypeptide.
 22. A method of delaying the onset ofrelapsing-remitting multiple sclerosis in a mammal comprisingadministering to the mammal a therapeutically effective amount of aformulation comprising a polypeptide encoded by a polynucleotide of SEQID NO:174 and a pharmaceutically acceptable vehicle, wherein the encodedpolypeptide comprises amino acid residues 1-176 of SEQ ID NO:175. 23.The method of claim 22 wherein the polypeptide is conjugated to apolyalkyl oxide moiety.
 24. The method of claim 23 wherein the polyalkyloxide moiety is polyethylene glycol.
 25. The method of claim 24 whereinthe polyethylene glycol is monomethoxy-PEG propionaldehyde.
 26. Themethod of claim 25 wherein the monomethoxy-PEG propionaldehyde has amolecular weight of about 20 Kd or 30 Kd.
 27. The method of claim 25wherein the monomethoxy-PEG propionaldehyde is linear or branched. 28.The method of claim 25 wherein the monomethoxy-PEG propionaldehyde isconjugated N-terminally to the polypeptide.